Detergent compositions comprising  xanthan lyase variants i

ABSTRACT

The present disclosure relates to detergent compositions comprising xanthan lyase variants and methods for use of said compositions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. National-Stage entry under 35 U.S.C. § 371based on International Application No. PCT/EP2017/071296, filed Aug. 24,2017, which was published under PCT Article 21(2) and which claimspriority to German Application No. 10 2016 215 868.6, filed Aug. 24,2016, which are all hereby incorporated in their entirety by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to detergent compositions, such aslaundry compositions and dish wash compositions, including hand wash andautomatic dish wash compositions, comprising novel xanthan lyasevariants exhibiting alterations relative to the parent xanthan lyase inone or more properties including: detergent stability (e.g. improvedstability in a detergent composition, e.g. in the presence of achelator, e.g. EDTA or citrate) and/or storage stability (e.g. improvedstorage stability in a detergent composition, e.g. in the presence of achelator, e.g. EDTA or citrate). The present disclosure further relatesto detergent compositions comprising novel xanthan lyase variants havingactivity on xanthan gum. The present disclosure also relates to methodsfor producing said detergent compositions and to the use of saiddetergent compositions in cleaning applications.

BACKGROUND

Xanthan gum is a polysaccharide derived from the bacterial coat ofXanthomonas campestris. It is produced by the fermentation of glucose,sucrose, or lactose by the Xanthomonas campestris bacterium. After afermentation period, the polysaccharide is precipitated from a growthmedium with isopropyl alcohol, dried, and ground into a fine powder.Later, it is added to a liquid medium to form the gum. Xanthan gum is anatural polysaccharide consisting of different sugars which areconnected by several different bonds, such asβ-D-mannosyl-β-D-1,4-glucuronosyl bonds andβ-D-glucosyl-β-D-1,4-glucosyl bonds. Xanthan gum is at least partlysoluble in water and forms highly viscous solutions or gels. Completeenzymatic degradation of xanthan gum requires several enzymaticactivities including xanthan lyase activity and endo-β-1,4-glucanaseactivity. Xanthan lyases are enzymes that cleave theβ-D-mannosyl-β-D-1,4-glucuronosyl bond of xanthan and have beendescribed in the literature. Xanthan lyases are known in the art, e.g.two xanthan lyases have been isolated from Paenibacillus alginolyticusXL-1 (e.g. Ruijssenaars et al. (1999) ‘A pyruvated mannose-specificxanthan lyase involved in xanthan degradation by Paenibacillusalginolyticus XL-1’, Appl. Environ. Microbiol. 65(6): 2446-2452, andRuijssenaars et al. (2000), ‘A novel gene encoding xanthan lyase ofPaenibacillus alginolyticus strain XL-1’, Appl. Environ. Microbiol.66(9): 3945-3950). Glycoside hydrolases are enzymes that catalyse thehydrolysis of the glycosyl bond to release smaller sugars. There areover 100 classes of glycoside hydrolases which have been classified, seeHenrissat et al. (1991) ‘A classification of glycosyl hydrolases basedon amino-acid sequence similarities’, J. Biochem. 280: 309-316 and theUniprot website at www.cazy.org. The glycoside hydrolase family 9 (GH9)includes over 70 different enzymes that are mostly endo-glucanases (EC3.2.1.4), cellobiohydrolases (EC 3.2.1.91), β-glucosidases (EC 3.2.1.21)and exo-β-glucosaminidase (EC 3.2.1.165). In recent years xanthan gumhas been used as an ingredient in many consumer products including foods(e.g. as thickening agent in salad dressings and dairy products) andcosmetics (e.g. as stabilizer and thickener in toothpaste and make-up,creams and lotions to prevent ingredients from separating and to providethe right texture of the product). Further, xanthan gum has found use inthe oil industry as an additive to regulate the viscosity of drillingfluids etc. The widespread use of xanthan gum has led to a desire todegrade solutions, gels or mixtures containing xanthan gum therebyallowing easier removal of the by-products. Xanthan lyases andendoglucanases for the degradation of xanthan gum and the use of suchenzymes for cleaning purposes, such as the removal of xanthan gumcontaining stains, and in the drilling and oil industries are known inthe art, e.g. WO2013167581A1.

The known xanthan lyase having SEQ ID NO: 2 was found to be sensitive tothe presence of detergents with chelators. To improve applicabilityand/or cost and/or the performance of such enzymes there is an ongoingsearch for variants with altered properties, such as increasedstability, e.g. improved stability in a detergent composition, e.g. inthe presence of a chelator, e.g. EDTA or citrate, etc. However,mutagenesis of large enzymes followed by purification and functionalanalysis of mutant libraries can be very expensive and laborious.

BRIEF SUMMARY

This disclosure provides a detergent composition including a xanthanlyase variant, comprising an alteration at one or more positions in atleast one chelator-induced instability region selected from the groupof:

-   -   i) region 3 corresponding to amino acids 731 to 803 of SEQ ID        NO:2,    -   ii) region 6 corresponding to amino acids 903 to 1004 of SEQ ID        NO: 2,    -   iii) region 1 corresponding to amino acids 154 to 176 of SEQ ID        NO: 2,    -   iv) region 2 corresponding to amino acids 614 to 658 of SEQ ID        NO: 2,    -   v) region 4 corresponding to amino acids 807 to 846 of SEQ ID        NO:2, and    -   vi) region 5 corresponding to amino acids 872 to 885 of SEQ ID        NO: 2,    -   wherein said variant has at least about 60% and less than about        100% sequence identity to SEQ ID NO: 2.

DETAILED DESCRIPTION

The following detailed description is merely exemplary in nature and isnot intended to limit the disclosure or the application and uses of thesubject matter as described herein. Furthermore, there is no intentionto be bound by any theory presented in the preceding background or thefollowing detailed description.

Since the known xanthan lyase having SEQ ID NO: 2 is a large enzyme(>1000 residues), it is difficult and expensive to randomly target itsproperties for improvement of, e.g., stability in a detergentcomposition, e.g., in the presence of a chelator.

In some aspects, the present disclosure identifies chelator-inducedinstability regions in the protein sequence/structure of the knownxanthan lyase having SEQ ID NO: 2 that are affected when the molecule isincubated in a buffer with EDTA, and therefore provides an importantguidance on where to mutate a xanthan lyase in order to stabilize themolecule in a detergent, e.g., detergent composition comprising achelator.

In some aspects, the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant, comprising an alteration(e.g., a substitution, deletion or insertion) at one or more positionsin a chelator-induced instability region selected from the group of:region 1 corresponding to amino acids 154 to 176 of SEQ ID NO: 2, region2 corresponding to amino acids 614 to 658 of SEQ ID NO: 2, region 3corresponding to amino acids 731 to 803 of SEQ ID NO: 2, region 4corresponding to amino acids 807 to 846 of SEQ ID NO: 2, region 5corresponding to amino acids 872 to 885 of SEQ ID NO: 2, and region 6corresponding to amino acids 903 to 1004 of SEQ ID NO: 2.

In some aspects, the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant, comprising an alteration(e.g., a substitution, deletion or insertion) at one or more positionsin a chelator-induced instability region selected from the group of:region 1 corresponding to amino acids 154 to 176 of SEQ ID NO: 2, region2 corresponding to amino acids 614 to 658 of SEQ ID NO: 2, region 3corresponding to amino acids 731 to 803 of SEQ ID NO: 2, region 4corresponding to amino acids 807 to 846 of SEQ ID NO: 2, region 5corresponding to amino acids 872 to 885 of SEQ ID NO: 2, and region 6corresponding to amino acids 903 to 1004 of SEQ ID NO: 2, wherein saidvariant has at least about 60% and less than about 100% sequenceidentity to SEQ ID NO: 2, preferably said xanthan lyase variant havingan activity on xanthan gum.

In some aspects, the present disclosure defines a chelator-inducedinstability region of a parent xanthan lyase (e.g., SEQ ID NO: 2) havingone or more of the following features: in the presence of a chelator isrelatively less conformationally stable than one or more or all of itsadjacent regions; and/or in the presence of a chelator is relativelymore exposed to said chelator than one or more or all of its adjacentregions; and/or in the presence of a chelator is relatively moreaccessible to said chelator than one or more or all of its adjacentregions; and/or in the presence of a chelator is relatively moreconformationally dynamic than one or more or all of its adjacentregions; and/or in the presence of a chelator is relatively morereceptive to deuterium incorporation than one or more or all of itsadjacent regions. In the present disclosure, when referring to“relatively” it means that the features of a given regio as indicatedabove are based on observed differences between its features and/orproperties in the presence of a chelator and its features and/orproperties in the absence of a chelator (i.e. in order to determine theimpact of a chelator, each region is compared to itself in the absenceof said chelator and any changes are determined relative to its nativefeatures/properties in the absence of a chelator) (see also Example 1).

In some aspects, the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having at least about61%, at least about 62%, at least about 63%, at least about 64%, atleast about 65%, at least about 66%, at least about 67%, at least about68%, at least about 69%, at least about 70%, at least about 71%, atleast about 72%, at least about 73%, at least about 74%, at least about75%, at least about 76%, at least about 77%, at least about 78%, atleast about 79%, at least about 80%, at least about 81%, at least about82%, at least about 83%, at least about 84%, at least about 85%, atleast about 86%, at least about 87%, at least about 88%, at least about89%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%sequence identity to SEQ ID NO: 2.

In some aspects, the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant comprising an alteration(e.g., a substitution, deletion or insertion) at one or more positionsin a region selected from the group of:

i) region 1 corresponding to amino acids 154 to 176 of SEQ ID NO: 2,e.g., said alteration at one or more positions selected from the groupof positions: 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, wherein saidpositions correspond to amino acid positions of SEQ ID NO: 2 (e.g.,using the numbering of SEQ ID NO: 2),

ii) region 2 corresponding to amino acids 614 to 658 of SEQ ID NO: 2,e.g., said alteration at one or more positions selected from the groupof positions: 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624,625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638,639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652,653, 654, 655, 656, 657, 658, wherein said positions correspond to aminoacid positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO:2),

iii) region 3 corresponding to amino acids 731 to 803 of SEQ ID NO: 2,e.g., said alteration at one or more positions selected from the groupof positions: 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741,742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755,756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769,770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783,784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797,798, 799, 800, 801, 802, 803, wherein said positions correspond to aminoacid positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO:2),

iv) region 4 corresponding to amino acids 807 to 846 of SEQ ID NO: 2,e.g., said alteration at one or more positions selected from the groupof positions: 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817,818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831,832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845,846, wherein said positions correspond to amino acid positions of SEQ IDNO: 2 (e.g., using the numbering of SEQ ID NO: 2),

v) region 5 corresponding to amino acids 872 to 885 of SEQ ID NO: 2,e.g., said alteration at one or more positions selected from the groupof positions: 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882,883, 884, 885, wherein said positions correspond to amino acid positionsof SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO: 2),

vi) region 6 corresponding to amino acids 903 to 1004 of SEQ ID NO: 2,e.g., said alteration at one or more positions selected from the groupof positions: 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913,914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927,928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941,942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955,956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969,970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983,984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997,998, 999, 1000, 1001, 1002, 1003, 1004, wherein said positionscorrespond to amino acid positions of SEQ ID NO: 2 (e.g., using thenumbering of SEQ ID NO: 2). In some aspects, the afore-mentionedvariants have at least about 60% and less than about 100% sequenceidentity to SEQ ID NO: 2, preferably said xanthan lyase variant havingan activity on xanthan gum.

In some aspects, the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant comprising an alteration(e.g., a substitution, deletion or insertion) at one or more positionsin two or more, three or more, four or more, five or all six regionsselected from the group of:

-   -   i) region 1 corresponding to amino acids 154 to 176 of SEQ ID        NO: 2,    -   ii) region 2 corresponding to amino acids 614 to 658 of SEQ ID        NO: 2,    -   iii) region 3 corresponding to amino acids 731 to 803 of SEQ ID        NO: 2,    -   iv) region 4 corresponding to amino acids 807 to 846 of SEQ ID        NO: 2,    -   v) region 5 corresponding to amino acids 872 to 885 of SEQ ID        NO: 2,    -   vi) region 6 corresponding to amino acids 903 to 1004 of SEQ ID        NO: 2.

In some aspects, the present disclosure relates to a detergentcomposition xanthan lyase variant having an alteration (e.g., asubstitution, deletion or insertion) at one or more positions selectedfrom the group of positions: 155, 159, 620, 624, 626, 631, 635, 645,649, 650, 656, 738, 745, 746, 748, 752, 753, 754, 757, 764, 769, 774,775, 777, 779, 782, 785, 786, 789, 792, 796, 799, 800, 801, 819, 824,843, 845, 875, 903, 911, 912, 915, 919, 921, 923, 925, 927, 928, 930,932, 933, 941, 966, 967, 991 and 998 of SEQ ID NO: 2.

In some aspects, the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having one or moresubstitutions selected from the group of: Y155E, A159P, K620R, A624E,A626G, T631N, T631E, S635E, S635T, S635Q, A645S, T649V, T649K, T649R,Q650G, I656V, G738L, K745R, F746L, L748T, P752R, P752K, G753E, G753Q,G753S, S754E, S754L, S754Q, S754R, S757D, S757P, S757E, P764V, P764K,A769D, A769T, A769R, A769S, A769E, A769Q, A769*, A774V, L775M, L775Y,L775A, L775I, L775S, L775F, L775Q, D777K, D777R, P779V, Y782I, A785T,N786K, G789R, K792W, K792Y, K792V, K792A, N796Q, A799H, V800P, D801G,K819R, K819T, K824R, A843P, D845E, 875T, K875E, T903A, T903Q, A911V,A911M, A911S, A912T, A912I, A912Y, T915Q, T915S, T915V, T915A, T919F,T919G, T919D, T921R, T921S, T923H, T923D, T925Q, T925D, T925R, T927K,D928W, Y930H, Y930L, Y930F, A932P, D933M, G941E, G941D, A966P, A967D,N991D and V998K.

In some aspects, the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having an alteration(e.g., a substitution, deletion or insertion) at one or more positionsselected from the group of: 624, 635, 649, 656, 738, 753, 754, 757, 769,775, 777, 801, 843 and 875.

In some aspects, the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having one or moresubstitutions selected from the group of: A624E, S635E, T649K, I656V,G738L, G753E, S754E, S754R, S757D, A769D, L775A, D777R, D801G, A843P andK875T.

In some aspects of the detergent compositions of the present disclosure,the xanthan lyase variant comprises an alteration at one or morepositions in at least one chelator-induced instability region as well asan alteration at one or more positions in at least one adjacent region.Thus, in some aspects the xanthan lyase variant, in addition to analteration in one or more positions in at least one region selected fromthe group of regions 1, 2, 3, 4, 5 and 6 as set forth above andelsewhere herein, further comprises an alteration (e.g., a substitution,deletion or insertion) at one or more positions in at least one regionselected from the group of:

-   -   vii) region 7 corresponding to amino acids 1 to 153 of SEQ ID        NO: 2,    -   viii) region 8 corresponding to amino acids 177 to 613 of SEQ ID        NO: 2,    -   ix) region 9 corresponding to amino acids 659 to 730 of SEQ ID        NO: 2,    -   x) region 10 corresponding to amino acids 804 to 806 of SEQ ID        NO: 2,    -   xi) region 11 corresponding to amino acids 847 to 871 of SEQ ID        NO: 2,    -   xii) region 12 corresponding to amino acids 886 to 902 of SEQ ID        NO: 2, and    -   xiii) region 13 corresponding to amino acids 1005 to 1037 of SEQ        ID NO: 2.

The xanthan lyase variant may e.g. comprise an alteration at one or morepositions in each of one or more, two or more, three or more, four ormore, five or more, six or all seven regions selected from the group ofregions 7, 8, 9, 10, 11, 12 and 13.

In some aspects, the alteration at one or more positions in at least oneregion selected from the group of regions 7, 8, 9, 10, 11, 12 and 13 isan alteration at one or more positions selected from the group of: 9,15, 18, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221,229, 234, 238, 240, 242, 243, 257, 258, 284, 291, 293, 316, 317, 320,324, 329, 333, 339, 341, 352, 354, 360, 372, 377, 399, 400, 419, 440,450, 451, 454, 458, 481, 492, 505, 533, 567, 568, 576, 578, 579, 582,664, 672, 703, 722, 726, 727, 728, 851, 855, 856, 867, 887, 892, 899,900, 901, 902, 915, 1008 and 1016 of SEQ ID NO: 2. The xanthan lyasevariant may e.g. comprise an alteration at two or more of thesepositions, e.g. at three, four, five, six, seven, eight, nine or ten ofthese positions.

In some aspects, the alteration at one or more positions in at least oneregion selected from the group of regions 7, 8, 9, 10, 11, 12 and 13comprises one or more substitutions selected from the group of: K9R,N15T, T18D, L46D, A58L, S66H, Q89Y, K95E, S100D, N106Y, Q109R, Q109D,Q109F, Q109K, Q109A, K183Q, K183R, V188I, A190Q, A203P, K204R, A221P,E229N, E229S, E229V, I234V, I238W, I238L, I238M, 1240W, N242S, G243V,Y257W, R258E, R284G, K291R, A293G, A293P, K316R, R317K, K320R, L324Q,K329R, K333R, L339M, I341P, V352I, S354P, K360G, K360R, Q372H, F377Y,N399K, K400R, F419Y, N440K, D450P, K451E, K451R, A454V, D458S, K481R,A492H, A492L, T5051, L533I, K567R, G568A, S578K, S578N, S578R, S579R,S579K, S582K, T664K, N672D, I703L, I722F, P726Q, T727P, M728V, S851F,K855R, E856D, P867S, K887R, N892Y, N892W, N892F, G899S, 1900G, D901A,T902F, N1008D and K1016T of SEQ ID NO: 2. The xanthan lyase variant maye.g. comprise two or more of these substitutions, e.g. three, four,five, six, seven, eight, nine or ten of said substitutions.

In some aspects of the detergent compositions of the present disclosure,the xanthan lyase variant comprises an alteration at one or morepositions in at least one region selected from the group of regions 1,2, 3, 4, 5 and 6, and an alteration at one or more positions in at leastone region selected from the group of regions 7, 8, 9, 10, 11, 12 and13. In one aspect, the variant comprises an alteration at one or morepositions selected from the group of positions 624, 631, 635, 649, 656,738, 752, 753, 754, 757, 769, 775, 777, 800, 801, 843, 875, 911 and 915,and an alteration at one or more positions selected from the group ofpositions 89, 100, 190, 229, 234, 352, 360, 399, 440, 458, 492, 567,582, 664, 672, 703, 728, 892, 1008 and 1016 of SEQ ID NO: 2.

The variant may, for example, comprise an alteration at two or morepositions, e.g. three, four, five or more positions, selected from thegroup of positions 624, 631, 635, 649, 656, 738, 752, 753, 754, 757,769, 775, 777, 800, 801, 843, 875, 911 and 915, and an alteration at twoor more positions, e.g. two, three, four, five or more positions,selected from the group of positions 89, 100, 190, 229, 234, 352, 360,399, 440, 458, 492, 567, 582, 664, 672, 703, 728, 892, 1008 and 1016 ofSEQ ID NO: 2.

Preferred positions for alteration in this aspect include one or morepositions selected from the group of positions 624, 635, 649, 656, 738,753, 754, 757, 769, 775, 777, 801, 843 and 875, and one or morepositions selected from the group of positions 100, 190, 229, 234, 360,399, 440, 458, 492, 567, 582, 672, 892 and 1008 of SEQ ID NO: 2.

In one embodiment of this aspect, the xanthan lyase variant comprisesone or more substitutions selected from the group of Q89Y, S100D, A190Q,E229S, I234V, V352I, K360G, N399K, N440K, D458S, A492H, A492L, K567R,S582K, T664K, N672D, I703L, M728V, N892Y N1008D and K1016T, and one ormore substitutions selected from the group of A624E, T631N, S635E,T649K, I656V, G738L, P752K, P752R, G753E, S754E, S754R, S757D, A769D,L775A, D777R, V800P, D801G, A843P, K875T, A911V and T915A. The variantmay, for example, comprise two or more substitutions, e.g. three, four,five or more substitutions, selected from the group of Q89Y, S100D,A190Q, E229S, I234V, V352I, K360G, N399K, N440K, D458S, A492H, A492L,K567R, S582K, T664K, N672D, I703L, M728V, N892Y N1008D and K1016T, andtwo or more substitutions, e.g. three, four, five or more substitutions,selected from the group of A624E, T631N, S635E, T649K, I656V, G738L,P752K, P752R, G753E, S754E, S754R, S757D, A769D, L775A, D777R, V800P,D801G, A843P, K875T, A911V and T915A. Preferred substitutions in thisembodiment include one or more substitutions selected from the group ofS100D, A190Q, E229S, I234V, K360G, N399K, N440K, D458S, A492H, K567R,S582K, N672D, N892Y and N1008D, and one or more substitutions selectedfrom the group of A624E, S635E, T649K, I656V, G738L, G753E, S754E,S754R, S757D, A769D, L775A, D777R, D801G, A843P and K875T.

Non-limiting examples of such variants include:

-   -   A190Q, E229S, S635E, T649K, I656V, N672D, I703L, G753E, S754E,        S757D, A769D, L775A, D801G, K875T, N892Y, N1008D    -   E229S, S635E, T649K, I656V, N672D, I703L, G753E, S754E, A769D,        L775A, D801G, K875T, N892Y, N1008D    -   E229S, V352I, S635E, T649K, I656V, N672D, G753E, S754E, A769D,        L775A, V800P, D801G, K875T, N892Y    -   E229S, K360G, D458S, S582K, N672D, G753E, S754E, A769D, L775A,        D801G, K875T, N892Y, N1008D    -   S100D, E229S, K360G, D458S, S582K, T664K, N672D, G753E, S754E,        S757D, A769D, L775A, D801G, A843P, K875T, N892Y, A911V, N1008D,        K1016T    -   E229S, I234V, S582K, N672D, G753E, S754E, A769D, L775A, V800P,        D801G, K875T, N892Y    -   Q89Y, E229S, N440K, S582K, A624E, N672D, G753E, S754E, A769D,        L775A, D801G, K875T, N892Y    -   E229S, S635E, T649K, I656V, N672D, P752K, G753E, A769D, L775A,        D801G, A843P, K875T, N892Y    -   E229S, S635E, T649K, I656V, N672D, G753E, S754E, S757D, A769D,        L775A, D801G, K875T, N892Y    -   E229S, N440K, S582K, N672D, G753E, S754E, A769D, L775A, D801G,        A843P, K875T, N892Y, N1008D    -   E229S, N440K, S582K, A624E, N672D, G753E, S754E, A769D, L775A,        V800P, D801G, K875T, N892Y    -   A190Q, E229S, S635E, T649K, I656V, N672D, P752K, G753E, A769D,        L775A, D801G, A843P, K875T, N892Y    -   A190Q, E229S, S582K, N672D, G753E, S754E, A769D, L775A, D801G,        K875T, N892Y    -   E229S, N440K, S582K, N672D, P752R, G753E, S754E, S757D, A769D,        L775A, D801G, K875T, N892Y, N1008D    -   E229S, S582K, S635E, N672D, P752R, G753E, S754E, A769D, L775A,        D801G, K875T, N892Y, N1008D    -   A190Q, E229S, N440K, S582K, A624E, S635E, N672D, G753E, S754E,        A769D, L775A, D801G, K875T, N892Y    -   E229S, I234V, A492L, S582K, N672D, G753E, S754E, A769D, L775A,        D801G, K875T, N892Y    -   A190Q, E229S, K360G, D458S, S582K, T664K, N672D, G753E, S754E,        A769D, L775A, D801G, K875T, N892Y, N1008D    -   S100D, E229S, K360G, D458S, S582K, N672D, G753E, S754E, S757D,        A769D, L775A, D801G, A843P, K875T, N892Y, T915A, N1008D    -   E229S, N440K, S582K, A624E, S635E, N672D, G738L, G753E, S754E,        S757D, A769D, L775A, D801G, K875T, N892Y    -   S100D, E229S, K360G, D458S, S582K, N672D, G753E, S754E, A769D,        L775A, D801G, K875T, N892Y, N1008D    -   A190Q, E229S, D458S, T631N, N672D, G753E, S754E, A769D, L775A,        D801G, A843P, K875T, N892Y    -   A190Q, E229S, K360G, D458S, S582K, N672D, G753E, S754E, A769D,        L775A, D801G, K875T, N892Y, N1008D    -   E229S, S635E, T649K, I656V, N672D, G753E, S754R, S757D, A769D,        L775A, D801G, A843P, K875T, N892Y    -   E229S, D458S, S582K, T631N, S635E, N672D, M728V, G753E, S754E,        S757D, A769D, L775A, D801G, K875T, N892Y    -   A190Q, E229S, K360G, D458S, S582K, N672D, G753E, S754E, S757D,        A769D, L775A, D801G, K875T, N892Y, N1008D    -   E229S, A492L, S635E, T649K, I656V, N672D, G753E, S757D, A769D,        L775A, D801G, K875T, N892Y    -   S100D, A190Q, E229S, K360G, D458S, S582K, N672D, G753E, S754E,        A769D, L775A, D801G, K875T, N892Y, N1008D    -   A190Q, E229S, I234V, S582K, N672D, G753E, S754E, S757D, A769D,        L775A, D801G, K875T, N892Y    -   E229S, N399K, D458S, A492H, K567R, S582K, S635E, T649K, N672D,        G753E, S754E, A769D, L775A, D777R, D801G, K875T, N892Y    -   E229S, D458S, A492L, T631N, N672D, G753E, S754E, S757D, A769D,        L775A, D801G, K875T, N892Y    -   E229S, D458S, A492H, K567R, S582K, S635E, N672D, G753E, S754E,        A769D, L775A, D777R, D801G, K875T, N892Y    -   S100D, E229S, K360G, D458S, S582K, N672D, G753E, S754E, S757D,        A769D, L775A, D801G, A843P, K875T, N892Y, N1008D    -   E229S, N399K, D458S, K567R, S582K, S635E, N672D, G753E, S754E,        A769D, L775A, D777R, D801G, K875T, N892Y.

In some aspects, the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having activity onxanthan gum; preferably said activity comprises xanthan lyase EC4.2.2.12 activity, further preferably said activity is xanthan lyase EC4.2.2.12 activity.

In some aspects, the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having an improvedstability in said detergent composition compared to a parent xanthanlyase (e.g., with SEQ ID NO: 2).

In some aspects, the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having a half-lifeimprovement factor (HIF) of >1.0 relative to a parent xanthan lyase.

In some aspects, the present disclosure relates to a detergentcomposition comprising an isolated xanthan lyase variant having activityon xanthan gum as contemplated herein. In a further aspect, thedetergent composition further comprises an isolated polypeptide havingGH9 endoglucanase activity.

In some aspects, a detergent composition of the present disclosurecomprises one or more detergent components for degrading xanthan gum.

In some aspects, the present disclosure relates to use of a detergentcomposition of the present disclosure, wherein said use is selected fromthe group of: use for degrading xanthan gum and use in a cleaningprocess, such as laundry or hard surface cleaning such as dish wash.

In some aspects, the present disclosure further relates to the use of adetergent composition of the present disclosure for degrading xanthangum or for washing or cleaning textiles and/or hard surfaces, such asdish wash, wherein the composition has an enzyme detergency benefit.

In some aspects, the present disclosure also relates to methods ofdegrading xanthan gum using detergent compositions of the presentdisclosure, wherein xanthan gum is on the surface of a hard surface ortextile.

Overview of Sequence Listing

SEQ ID NO: 1 is the DNA sequence of the parent mature xanthan lyase froma strain of a Paenibacillus sp.

SEQ ID NO: 2 is the amino acid sequence of the mature polypeptideencoded by SEQ ID NO: 1.

Definitions

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Cleaning or Detergent Application: the term “cleaning or detergentapplication” means applying the xanthan lyase of the application in anycomposition for the purpose of cleaning or washing, by hand, machine orautomated, a hard surface or a textile.

Cleaning Composition: the term “cleaning composition” refers tocompositions that find use in the removal of undesired compounds fromitems to be cleaned, such as textiles, dishes, and hard surfaces. Theterms encompass any materials/compounds selected for the particular typeof cleaning composition desired and the form of the product (e.g.,liquid, gel, powder, granulate, paste, or spray compositions) andincludes, but is not limited to, detergent compositions (e.g., liquidand/or solid laundry detergents and fine fabric detergents; hard surfacecleaning formulations, such as for glass, wood, ceramic and metalcounter tops and windows; carpet cleaners; oven cleaners; fabricfresheners; fabric softeners; and textile and laundry pre-spotters, aswell as dish wash detergents). In addition to the xanthan lyase, thedetergent formulation may contain one or more additional enzymes (suchas xanthan lyases, proteases, amylases, lipases, cutinases, cellulases,xanthan lyases, xyloglucanases, pectinases, pectin lyases, xanthanases,peroxidaes, haloperoxygenases, catalases and mannanases, or any mixturethereof), and/or components such as surfactants, builders, chelators orchelating agents, bleach system or bleach components, polymers, fabricconditioners, foam boosters, suds suppressors, dyes, perfume, tannishinhibitors, optical brighteners, bactericides, fungicides, soilsuspending agents, anti corrosion agents, enzyme inhibitors orstabilizers, enzyme activators, transferase(s), hydrolytic enzymes,oxido reductases, bluing agents and fluorescent dyes, antioxidants, andsolubilizers.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Colour clarification: During washing and wearing loose or broken fiberscan accumulate on the surface of the fabrics. One consequence can bethat the colours of the fabric appear less bright or less intensebecause of the surface contaminations. Removal of the loose or brokenfibers from the textile will partly restore the original colours andlooks of the textile. By the term “colour clarification”, as usedherein, is meant the partial restoration of the initial colours oftextile.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present disclosure. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Corresponding to: The term “corresponding to” as used herein, refers toa way of determining the specific amino acid of a sequence whereinreference is made to a specific amino acid sequence. E.g. for thepurposes of the present disclosure, when references are made to specificamino acid positions, the skilled person would be able to align anotheramino acid sequence to said amino acid sequence that reference has beenmade to, in order to determine which specific amino acid may be ofinterest in said another amino acid sequence. Alignment of another aminoacid sequence with e.g. the sequence as set forth in SEQ ID NO: 2, orany other amino acid sequence listed herein, has been describedelsewhere herein. Alternative alignment methods may be used, and arewell-known for the skilled person.

Degrading xanthan gum and xanthan gum degrading activity: The terms“degrading xanthan gum” and “xanthan gum degrading activity” are usedinterchangeably and are defined as the depolymerisation, degradation orbreaking down of xanthan gum into smaller components. The degradation ofxanthan gum can either be the removal of one or more side chainsaccharides, the cutting of the backbone of xanthan gum into smallercomponents or the removal of one or more side chain saccharides and thecutting of the backbone of xanthan gum into smaller components. Apreferred assay for measuring degradation of xanthan gum is described inexample 4 herein. Non-limiting examples of the xanthan gum degradingactivity include xanthan lyase EC 4.2.2.12 activity.

Detergent component: the term “detergent component” is defined herein tomean the types of chemicals which can be used in detergent compositions.Examples of detergent components are surfactants, hydrotropes, builders,co-builders, chelators or chelating agents, bleaching system or bleachcomponents, polymers, fabric hueing agents, fabric conditioners, foamboosters, suds suppressors, dispersants, dye transfer inhibitors,fluorescent whitening agents, perfume, optical brighteners,bactericides, fungicides, soil suspending agents, soil release polymers,anti-redeposition agents, enzyme inhibitors or stabilizers, enzymeactivators, antioxidants, and solubilizers. The detergent compositionmay comprise of one or more of any type of detergent component.

Detergent composition: the term “detergent composition” refers tocompositions that find use in the removal of undesired compounds fromitems to be cleaned, such as textiles, dishes, and hard surfaces. Thedetergent composition may be used to e.g. clean textiles, dishes andhard surfaces for both household cleaning and industrial cleaning. Theterms encompass any materials/compounds selected for the particular typeof cleaning composition desired and the form of the product (e.g.,liquid, gel, powder, granulate, paste, or spray compositions) andincludes, but is not limited to, detergent compositions (e.g., liquidand/or solid laundry detergents and fine fabric detergents; hard surfacecleaning formulations, such as for glass, wood, ceramic and metalcounter tops and windows; carpet cleaners; oven cleaners; fabricfresheners; fabric softeners; and textile and laundry pre-spotters, aswell as dish wash detergents). In addition to containing a xanthan lyaseof the present disclosure and/or a GH9 endoglucanase, the detergentformulation may contain one or more additional enzymes (such asendoglucanases, xanthan lyases, proteases, amylases, lichenases,lipases, cutinases, cellulases, xanthan lyases, xyloglucanases,pectinases, pectin lyases, xanthanases, peroxidaes, haloperoxygenases,catalases and mannanases, or any mixture thereof), and/or componentssuch as surfactants, builders, chelators or chelating agents, bleachsystem or bleach components, polymers, fabric conditioners, foamboosters, suds suppressors, dyes, perfume, tannish inhibitors, opticalbrighteners, bactericides, fungicides, soil suspending agents,anti-corrosion agents, enzyme inhibitors or stabilizers, enzymeactivators, transferase(s), hydrolytic enzymes, oxido reductases, bluingagents and fluorescent dyes, antioxidants, and solubilizers.

Dish wash: The term “dish wash” refers to all forms of washing dishes,e.g. by hand or automatic dish wash. Washing dishes includes, but is notlimited to, the cleaning of all forms of crockery such as plates, cups,glasses, bowls, all forms of cutlery such as spoons, knives, forks andserving utensils as well as ceramics, plastics, metals, china, glass andacrylics.

Dish washing composition: The term “dish washing composition” refers toall forms of compositions for cleaning hard surfaces. The presentdisclosure is not restricted to any particular type of dish washcomposition or any particular detergent.

Endoglucanase: The term “endoglucanase” or “EG” means an endo-1,4- orendo-1,3;1,4-beta-D-glucan 4-glucanohydrolase (e.g., EC 3.2.1.4) thatcatalyses endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3/beta-1,4 glucanssuch as cereal beta-D-glucans, xyloglucans, xanthans and other plantmaterial containing cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481).

Enzyme detergency benefit: The term “enzyme detergency benefit” isdefined herein as the advantageous effect an enzyme may add to adetergent compared to the same detergent without the enzyme. Importantdetergency benefits which can be provided by enzymes are stain removalwith no or very little visible soils after washing and or cleaning,prevention or reduction of redeposition of soils released in the washingprocess an effect that also is termed anti-redeposition, restoring fullyor partly the whiteness of textiles, which originally were white butafter repeated use and wash have obtained a greyish or yellowishappearance an effect that also is termed whitening. Textile carebenefits, which are not directly related to catalytic stain removal orprevention of redeposition of soils are also important for enzymedetergency benefits. Examples of such textile care benefits areprevention or reduction of dye transfer from one fabric to anotherfabric or another part of the same fabric an effect that is also termeddye transfer inhibition or anti-backstaining, removal of protruding orbroken fibers from a fabric surface to decrease pilling tendencies orremove already existing pills or fuzz an effect that also is termedanti-pilling, improvement of the fabric-softness, colour clarificationof the fabric and removal of particulate soils which are trapped in thefibers of the fabric or garment. Enzymatic bleaching is a further enzymedetergency benefit where the catalytic activity generally is used tocatalyze the formation of bleaching component such as hydrogen peroxideor other peroxides.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide having one or moreamino acids absent from the amino and/or carboxyl terminus of a maturepolypeptide; wherein the fragment has xanthan lyase activity. In oneaspect, a fragment contains at least about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94% or about 95% of the number of amino acids of the mature polypeptide.

Endoglucanase variant having activity on xanthan gum pre-treated withxanthan lyase: The term “Endoglucanase variant having activity onxanthan gum pre-treated with xanthan lyase” or an “endoglucanase havingactivity on xanthan gum pre-treated with xanthan lyase and belonging tothe GH9 class of glycosyl hydrioases” is defined as a polypeptidecomprising a domain belonging to the GH9 class of glycosyl hydrolases,and having activity (e.g., enzymatic activity, xanthan degradingactivity, endoglucanase EC 3.2.1.4 activity) on xanthan gum pre-treatedwith xanthan lyase.

Xanthan lyase variant having activity on xanthan gum: The term “Xanthanlyase variant having activity on xanthan gum” is defined as apolypeptide that cleaves the β-D-mannosyl-β-D-1,4-glucuronosyl bond ofxanthan (e.g., xanthan lyase EC 4.2.2.12 activity). A preferred assayfor measuring activity on xanthan gum is disclosed in example 4 herein.Examples of the xanthan lyase variants having activity on xanthan gum,are xanthan lyase polypeptides as such. Thus, polypeptides that thatcleaves the β-D-mannosyl-β-D-1,4-glucuronosyl bond of xanthan.

Half-life: The term “half-life” refers to the time it takes for anenzyme to lose half of its enzymatic activity under a given set ofconditions. It is denoted as T_(1/2) and is measured in hours (hrs).Half-life: can be calculated at a given detergent concentration andstorage temperature for parent (e.g. wild-type) and/or variants, as thedegradation follows an exponential decay and the incubation time (hours)is known, i.e. according to the following formulas:

T½(variant)=(Ln(0.5)/Ln(RA−variant/100))*Time

T½(wild-type)=(Ln(0.5)/Ln(RA−wild-type/100))*Time

Where ‘RA’ is the residual activity in percent and ‘Time’ is theincubation time in hours.

Half-life improvement factor: The term “Half-life improvement factor” or“HIF” is the improvement of half-life of a variant compared to theparent polypeptide, such as the parent xanthan lyase. A half-lifeimprovement factor (HIF) under a given set of storage conditions(detergent concentration and temperature) can be calculated as

${HIF} = \frac{{T\; {1/2}},{variant}}{{T\; {1/2}},{wt}}$

Where the wild-type (wt) is incubated under the same storage conditions(detergent concentration and incubation temperature) as the variant. Inthe cases where the difference in stability between wild-type andvariant is too big to accurately assess half-life for both wild-type andvariant using the same incubation time, the incubation time forwild-type and variant is different e.g. about 1 hr for wild-type andabout 840 hrs for the most stable variants.

Hard surface cleaning: The term “hard surface cleaning” is definedherein as cleaning of hard surfaces wherein hard surfaces may includefloors, tables, walls, roofs etc. as well as surfaces of hard objectssuch as cars (car wash) and dishes (dish wash). Dish washing includesbut are not limited to cleaning of plates, cups, glasses, bowls, andcutlery such as spoons, knives, forks, serving utensils, ceramics,plastics, metals, china, glass and acrylics.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present disclosure. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchimproved properties include, but are not limited to, catalyticefficiency, catalytic rate, chemical stability, oxidation stability, pHactivity, pH stability, specific activity, stability under storageconditions, chelator stability, substrate binding, substrate cleavage,substrate specificity, substrate stability, surface properties, thermalactivity, and thermostability.

Improved wash performance: The term “improved wash performance” isdefined herein as a (variant) enzyme (also a blend of enzymes, notnecessarily only variants but also backbones, and in combination withcertain cleaning composition etc.) displaying an alteration of the washperformance of a protease variant relative to the wash performance ofthe parent protease variant e.g. by increased stain removal. The term“wash performance” includes wash performance in laundry but also e.g. indish wash.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Laundering: The term “laundering” relates to both household launderingand industrial laundering and means the process of treating textileswith a solution containing a cleaning or detergent composition of thepresent disclosure. The laundering process can for example be carriedout using e.g. a household or an industrial washing machine or can becarried out by hand.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 1037 of SEQ ID NO: 2.

It is known in the art that a host cell may produce a mixture of two ofmore different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide. Itis also known in the art that different host cells process polypeptidesdifferently, and thus, one host cell expressing a polynucleotide mayproduce a different mature polypeptide (e.g., having a differentC-terminal and/or N-terminal amino acid) as compared to another hostcell expressing the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving enzymatic activity such as activity on xanthan gum pre-treatedwith xanthan lyase or xanthan lyase activity. In one aspect, the maturepolypeptide coding sequence is nucleotides 1 to 3111 of SEQ ID NO: 1.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent: The term “parent” or “parent xanthan lyase” means anypolypeptide with xanthan lyase activity to which an alteration is madeto produce the enzyme variants of the present disclosure. In one aspect,the parent is a xanthan lyase having the identical amino acid sequenceof the variant, but not having the alterations at one or more of thespecified positions. It will be understood, that the expression “havingidentical amino acid sequence” relates to about 100% sequence identity.Non-limiting examples of parent xanthan lyases include the mature parentxanthan lyase having SEQ ID NO: 2.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”. For purposes of the present disclosure, the sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J Mol. Biol. 48:443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 orlater. The parameters used are gap open penalty of about 10, gapextension penalty of about 0.5, and the EBLOSUM62 (EMBOSS version ofBLOSUM62) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present disclosure, the sequence identity betweentwo deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 5.0.0 or later. The parameters used are gapopen penalty of about 10, gap extension penalty of about 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labelled “longest identity” (obtained using the—nobriefoption) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Stringency conditions: The different stringency conditions are definedas follows.

The term “very low stringency conditions” means for probes of at leastabout 100 nucleotides in length, prehybridization and hybridization atabout 42° C. in 5×SSPE, about 0.3% SDS, about 200 micrograms/ml shearedand denatured salmon sperm DNA, and about 25% formamide, followingstandard Southern blotting procedures for from about 12 to about 24hours. The carrier material is finally washed three times each for about15 minutes using 2×SSC, about 0.2% SDS at about 45° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at about 42°C. in 5×SSPE, about 0.3% SDS, about 200 micrograms/ml sheared anddenatured salmon sperm DNA, and about 25% formamide, following standardSouthern blotting procedures for from about 12 to about 24 hours. Thecarrier material is finally washed three times each for about 15 minutesusing 2×SSC, from about 0.2% SDS at about 50° C.

The term “medium stringency conditions” means for probes of at leastabout 100 nucleotides in length, prehybridization and hybridization atabout 42° C. in 5×SSPE, about 0.3% SDS, about 200 micrograms/ml shearedand denatured salmon sperm DNA, and 35% formamide, following standardSouthern blotting procedures for from about 12 to about 24 hours. Thecarrier material is finally washed three times each for about 15 minutesusing 2×SSC, about 0.2% SDS at about 55° C.

The term “medium-high stringency conditions” means for probes of atleast about 100 nucleotides in length, prehybridization andhybridization at about 42° C. in 5×SSPE, about 0.3% SDS, about 200micrograms/ml sheared and denatured salmon sperm DNA, and about 35%formamide, following standard Southern blotting procedures for fromabout 12 to about 24 hours. The carrier material is finally washed threetimes each for about 15 minutes using 2×SSC, about 0.2% SDS at about 60°C.

The term “high stringency conditions” means for probes of at least about100 nucleotides in length, prehybridization and hybridization at about42° C. in 5×SSPE, about 0.3% SDS, about 200 micrograms/ml sheared anddenatured salmon sperm DNA, and about 50% formamide, following standardSouthern blotting procedures for from about 12 to about 24 hours. Thecarrier material is finally washed three times each for about 15 minutesusing 2×SSC, about 0.2% SDS at about 65° C.

The term “very high stringency conditions” means for probes of at leastabout 100 nucleotides in length, prehybridization and hybridization atabout 42° C. in 5×SSPE, about 0.3% SDS, about 200 micrograms/ml shearedand denatured salmon sperm DNA, and about 50% formamide, followingstandard Southern blotting procedures for from about 12 to about 24hours. The carrier material is finally washed three times each for about15 minutes using 2×SSC, about 0.2% SDS at about 70° C.

Subsequence: The term “subsequence” means a polynucleotide having one ormore nucleotides absent from the 5′ and/or 3′ end of a maturepolypeptide coding sequence; wherein the subsequence encodes a fragmenthaving enzymatic activity, such as activity on xanthan gum pre-treatedwith xanthan lyase or xanthan lyase activity.

Textile: The term “textile” means any textile material including yarns,yarn intermediates, fibers, non-woven materials, natural materials,synthetic materials, and any other textile material, fabrics made ofthese materials and products made from fabrics (e.g., garments and otherarticles). The textile or fabric may be in the form of knits, wovens,denims, non-wovens, felts, yarns, and towelling. The textile may becellulose based such as natural cellulosics, including cotton,flax/linen, jute, ramie, sisal or coir or manmade cellulosics (e.g.originating from wood pulp) including viscose/rayon, ramie, celluloseacetate fibers (tricell), lyocell or blends thereof. The textile orfabric may also be non-cellulose based such as natural polyamidesincluding wool, camel, cashmere, mohair, rabit and silk or syntheticpolymer such as nylon, aramid, polyester, acrylic, polypropylen andspandex/elastane, or blends thereof as well as blend of cellulose basedand non-cellulose based fibers. Examples of blends are blends of cottonand/or rayon/viscose with one or more companion material such as wool,synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyesterfibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyurethane fibers, polyurea fibers, aramid fibers), andcellulose-containing fibers (e.g. rayon/viscose, ramie, flax/linen,jute, cellulose acetate fibers, lyocell). Fabric may be conventionalwashable laundry, for example stained household laundry. When the termfabric or garment is used it is intended to include the broader termtextiles as well.

Textile care benefit: “Textile care benefits”, which are not directlyrelated to catalytic stain removal or prevention of redeposition ofsoils, are also important for enzyme detergency benefits. Examples ofsuch textile care benefits are prevention or reduction of dye transferfrom one textile to another textile or another part of the same textilean effect that is also termed dye transfer inhibition oranti-backstaining, removal of protruding or broken fibers from a textilesurface to decrease pilling tendencies or remove already existing pillsor fuzz an effect that also is termed anti-pilling, improvement of thetextile-softness, colour clarification of the textile and removal ofparticulate soils which are trapped in the fibers of the textile.Enzymatic bleaching is a further enzyme detergency benefit where thecatalytic activity generally is used to catalyse the formation ofbleaching component such as hydrogen peroxide or other peroxides orother bleaching species.

Variant: The term “variant” means a polypeptide (e.g., a xanthan lyasepolypeptide) comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding one or more amino acids, e.g.,1-5 amino acids adjacent to and immediately following the amino acidoccupying a position. Non-limiting examples of xanthan lyase variants ofthe present disclosure include xanthan lyase variants having an activityon xanthan gum. Non-limiting examples of variants of the presentdisclosure further include variants having at least about 20%, e.g., atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95%, or atleast about 100% xanthan lyase activity of the mature parent xanthanlyase of SEQ ID NO: 2. A preferred assay for measuring activity onxanthan gum is disclosed in example 4 herein.

Stability: The term “stability” means resistance or the degree ofresistance to change, unfolding, disintegration, denaturation oractivity loss. Non-limiting examples of stability include conformationalstability, storage stability and stability during use, e.g. during awash process and reflects the stability of a polypeptide (e.g. a xanthanlyase variant as contemplated herein) as a function of time, e.g. howmuch activity is retained when said polypeptide (e.g. said xanthan lyasevariant) is kept in solution, in particular in a detergent solution. Thestability is influenced by many factors, e.g. presence of chelator(s),pH, temperature, detergent composition, e.g. amount of builder(s),surfactant(s), chelator(s) etc. The xanthan lyase stability may bemeasured using a half-life improvement factor (HIF) as described inexample 4 herein.

Improved stability: The term “improved stability” or “increasedstability” is defined herein as increased stability in a detergentcomposition (e.g., in solutions, e.g. in the presence of a chelator,e.g. EDTA or citrate), relative to the stability of the parent xanthanlyase, relative to a xanthan lyase having the identical amino acidsequence of the variant, but not having the alterations at one or moreof the specified positions, or relative to SEQ ID NO: 2. The terms“improved stability” and “increased stability” includes “improvedchemical stability”, “detergent stability” and “improved detergentstability.

Improved chemical stability: The term “improved chemical stability” isdefined herein as a variant enzyme displaying retention of enzymaticactivity after a period of incubation in the presence of a chemical orchemicals, either naturally occurring or synthetic, which reduces theenzymatic activity of the parent enzyme. Improved chemical stability mayalso result in variants being more able (e.g., better that the parent)to catalyze a reaction in the presence of such chemicals. In aparticular aspect of the present disclosure the improved chemicalstability is an improved stability in a detergent, in particular in aliquid detergent. The term “detergent stability” or “improved detergentstability is in particular an improved stability of the xanthan lyasecompared to the parent xanthan lyase, when a xanthan lyase variant ofthe present disclosure is mixed into a liquid detergent formulation,especially into a liquid detergent formulation comprising a chelator(e.g. EDTA or citrate).

Conformational stability: The term “conformational stability” means aresistance or a degree of resistance to conformational change, unfoldingor disintegration. Accordingly, the term “less conformationally stable”means less resistant or having lesser degree of resistance toconformational change, unfolding or disintegration.

Instability: The term “instability” means lack of stability.Non-limiting examples of instability include conformational instability,unfolding, denaturation, disintegration, activity loss.

Chelator-induced instability region: The term “chelator-inducedinstability region” means any region of a polypeptide contributing toinstability of said polypeptide in the presence of a chelator.Non-limiting examples of chelators include EDTA(Ethylenediaminetetraacetic acid) and citrate. Non-limiting examples ofchelator-induced instability regions include any region of a polypeptidehaving one or more of the following features: in the presence of achelator it is less conformationally stable than one or more or all ofits adjacent regions; and/or in the presence of a chelator it is moreexposed to said chelator than one or more or all of its adjacentregions; and/or in the presence of a chelator it is more accessible tosaid chelator than one or more or all of its adjacent regions; and/or inthe presence of a chelator it is more conformationally dynamic than oneor more or all of its adjacent regions; and/or in the presence of achelator it is more receptive to deuterium incorporation than one ormore or all of its adjacent regions. Non-limiting examples ofchelator-induced instability regions further include any region of apolypeptide responsible for chelator-induced instability. Non-limitingexamples of chelator-induced instability regions of a mature xanthanlyase (e.g. having SEQ ID NO: 2) include: region 1 corresponding toamino acids 154 to 176 of SEQ ID NO: 2, region 2 corresponding to aminoacids 614 to 658 of SEQ ID NO: 2, region 3 corresponding to amino acids731 to 803 of SEQ ID NO: 2, region 4 corresponding to amino acids 807 to846 of SEQ ID NO: 2, region 5 corresponding to amino acids 872 to 885 ofSEQ ID NO: 2, and region 6 corresponding to amino acids 903 to 1004 ofSEQ ID NO: 2. Non-limiting examples of regions adjacent tochelator-induced instability regions of a mature xanthan lyase (e.g.having SEQ ID NO: 2) include: region 7 corresponding to amino acids 1 to153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 ofSEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ IDNO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO:2, region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2,region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, andregion 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2.

Adjacent region: The term “adjacent region” means any region of apolypeptide that is not a chelator-induced instability region.Non-limiting examples of adjacent regions of a mature xanthan lyase(e.g. having SEQ ID NO: 2) include: region 7 corresponding to aminoacids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806of SEQ ID NO: 2, region 11 corresponding to amino acids 847 to 871 ofSEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQID NO: 2.

Chelator exposure: The term “chelator exposure” means concentration oramount of a chelator that reaches a polypeptide. Accordingly, in thecontext of the present disclosure the term “more exposed to a chelator”means that chelator exposure of a particular region (e.g. achelator-induced instability region) is greater than a chelator exposureof a different region (e.g. an adjacent region). In one aspect, chelatorexposure can be expressed in numerical terms of concentration, duration,and frequency (e.g. for chemical agents, e.g. chelators) or intensity.

Chelator accessibility: The term “chelator accessibility” encompassesopenness to the influence by a chelator and easiness of approach bychelator. Accordingly, in the context of the present disclosure the term“more accessible to a chelator” means that chelator accessibility of aparticular region (e.g. a chelator-induced instability region) isgreater than a chelator accessibility of a different region (e.g. anadjacent region).

Conformational dynamics: The term “conformational dynamics” encompassesvibrations, structural rearrangements and transitions of a polypeptide(e.g. in solution). Accordingly, in the context of the presentdisclosure the term “more conformationally dynamic” means thatconformational dynamics of a particular region (e.g. a chelator-inducedinstability region) is greater than conformational dynamics of adifferent region (e.g. an adjacent region).

Receptiveness to deuterium incorporation: The term “receptiveness todeuterium incorporation” means amount of hydrogen atoms replaced by adeuterium atoms during hydrogen-deuterium exchange. Said amount can bemeasured in relative (e.g. compared to another amount) or absolute (e.g.expressed numerically) terms. Accordingly, in the context of the presentdisclosure the term “more receptive to deuterium incorporation” meansthat receptiveness to deuterium incorporation of a particular region(e.g. a chelator-induced instability region) is greater thanreceptiveness to deuterium incorporation of a different region (e.g. anadjacent region).

Wash performance: The term “wash performance” is used as an enzyme'sability to remove stains present on the object to be cleaned during e.g.wash or hard surface cleaning. The improvement in the wash performancemay be quantified by calculating the so-called intensity value (Int) in‘Automatic Mechanical Stress Assay (AMSA) for laundry’ or the remissionvalue (Rem).

Conventions for Designation of Variants

For purposes of the present disclosure, the mature polypeptide disclosedin SEQ ID NO: 2 is used to determine the corresponding amino acidresidue in another xanthan lyase. The amino acid sequence of anotherxanthan lyase is aligned with the mature polypeptide disclosed in SEQ IDNO: 2, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed in SEQ ID NO: 2 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 5.0.0 or later. Theparameters used are gap open penalty of about 10, gap extension penaltyof about 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix.

Identification of the corresponding amino acid residue in anotherxanthan lyase can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 2 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present disclosure, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions.

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of threonine at position 226 with alanine is designated as“Thr226Ala” or “T226A”. Multiple mutations are separated by additionmarks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”, representingsubstitutions at positions 205 and 411 of glycine (G) with arginine (R)and serine (S) with phenylalanine (F), respectively.

Deletions.

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position, *. Accordingly, the deletion of glycine atposition 195 is designated as “Gly195*” or “G195*”. Multiple deletionsare separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or“G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used:Original amino acid, position, original amino acid, inserted amino acid.Accordingly the insertion of lysine after glycine at position 195 isdesignated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”. An indication of aninsertion at a particular position is understood as being an insertionafter the original amino acid residue. For example, an “insertion atposition 195” is understood to be an insertion after the originalresidue in position 195.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G-K-A

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks(“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing asubstitution of arginine and glycine at positions 170 and 195 withtyrosine and glutamic acid, respectively.

Different Alterations.

Where different alterations can be introduced at a position, thedifferent alterations are separated by a comma, e.g., “Arg170Tyr,Glu”represents a substitution of arginine at position 170 with tyrosine orglutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates thefollowing variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”,“Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

Alternatively, different alterations or may be indicated using brackets,e.g., Arg170[Tyr, Gly] or in one-letter code R170 [Y,G].

The known xanthan lyase having SEQ ID NO: 2 is a large enzyme (> about1000 residues), it is therefore extremely laborious and expensive totarget its properties for improvement of, e.g., stability in a detergentcomposition, e.g. in the presence of a chelator. In some aspects, thepresent disclosure narrows down the number of residues to target whentrying to stabilize xanthan lyase molecules using protein engineering toa region selected from the group of: region 1 corresponding to aminoacids 154 to 176 of SEQ ID NO: 2, region 2 corresponding to amino acids614 to 658 of SEQ ID NO: 2, region 3 corresponding to amino acids 731 to803 of SEQ ID NO: 2, region 4 corresponding to amino acids 807 to 846 ofSEQ ID NO: 2, region 5 corresponding to amino acids 872 to 885 of SEQ IDNO: 2, and region 6 corresponding to amino acids 903 to 1004 of SEQ IDNO: 2.

In one embodiment the present disclosure dramatically narrows down thenumber of residues to target when trying to stabilize xanthan lyasemolecules using protein engineering.

Variants

In one embodiment, the chelator-induced instability regions in theprotein sequence of the known xanthan lyase having SEQ ID NO: 2 that areaffected when the molecule is incubated in a buffer with EDTA, are thefollowing: region 1 corresponding to amino acids 154 to 176 of SEQ IDNO: 2, region 2 corresponding to amino acids 614 to 658 of SEQ ID NO: 2,region 3 corresponding to amino acids 731 to 803 of SEQ ID NO: 2, region4 corresponding to amino acids 807 to 846 of SEQ ID NO: 2, region 5corresponding to amino acids 872 to 885 of SEQ ID NO: 2, and region 6corresponding to amino acids 903 to 1004 of SEQ ID NO: 2. This relatesto an important guidance on where to mutate a xanthan lyase in order tostabilize the molecule in a detergent, e.g. detergent compositioncomprising a chelator, e.g. EDTA or citrate.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant, comprising an alteration(e.g., a substitution, deletion or insertion) at one or more positionsin a region selected from the group of: region 1 corresponding to aminoacids 154 to 176 of SEQ ID NO: 2, region 2 corresponding to amino acids614 to 658 of SEQ ID NO: 2, region 3 corresponding to amino acids 731 to803 of SEQ ID NO: 2, region 4 corresponding to amino acids 807 to 846 ofSEQ ID NO: 2, region 5 corresponding to amino acids 872 to 885 of SEQ IDNO: 2, and region 6 corresponding to amino acids 903 to 1004 of SEQ IDNO: 2, wherein said variant has at least 60% and less than 100% sequenceidentity to SEQ ID NO: 2; preferably said xanthan lyase variant hasactivity on xanthan gum, further preferably said activity is a xanthangum degrading activity.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant, comprising an alteration(e.g., a substitution, deletion or insertion) at one or more positionsin two or more regions selected from the group of: region 1corresponding to amino acids 154 to 176 of SEQ ID NO: 2, region 2corresponding to amino acids 614 to 658 of SEQ ID NO: 2, region 3corresponding to amino acids 731 to 803 of SEQ ID NO: 2, region 4corresponding to amino acids 807 to 846 of SEQ ID NO: 2, region 5corresponding to amino acids 872 to 885 of SEQ ID NO: 2, and region 6corresponding to amino acids 903 to 1004 of SEQ ID NO: 2, wherein saidvariant has at least about 60% and less than about 100% sequenceidentity to SEQ ID NO: 2; preferably said xanthan lyase variant hasactivity on xanthan gum, further preferably said activity is a xanthangum degrading activity.

In one embodiment the present disclosure relates to a detergentcomposition comprising a parent xanthan lyase as described herein (e.g.,SEQ ID NO: 2) having an alteration (e.g., a substitution, deletion orinsertion) at one or more positions in a region selected from the groupof regions 1-6, wherein said region is a chelator-induced instabilityregion, preferably said chelator-induced instability region has one ormore of the following features: in the presence of a chelator it is lessconformationally stable than one or more or all of its adjacent regions;and/or in the presence of a chelator it is more exposed to said chelatorthan one or more or all of its adjacent regions; and/or in the presenceof a chelator it is more accessible to said chelator than one or more orall of its adjacent regions; and/or in the presence of a chelator it ismore conformationally dynamic than one or more or all of its adjacentregions; and/or in the presence of a chelator it is more receptive todeuterium incorporation than one or more or all of its adjacent regions;further preferably said adjacent region is selected from the group of:region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 11corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, further mostpreferably said chelator is EDTA or citrate.

In one embodiment the adjacent regions can be one or more or all of thefollowing: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO:2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2,region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 11corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having an alteration(e.g., a substitution, deletion or insertion) at one or more positionsin a region selected from the group of regions 1-6 (e.g., of SEQ ID NO:2 or another parent xanthan lyase), wherein in an aqueous solutioncomprising a detergent component said region (e.g., of SEQ ID NO: 2 oranother parent xanthan lyase) is relatively more accessible to saiddetergent component than one or more or all of its adjacent regions.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having an alteration(e.g., a substitution, deletion or insertion) at one or more positionsin a region selected from the group of regions 1-6 (e.g., of SEQ ID NO:2 or another parent xanthan lyase), wherein in an aqueous solutioncomprising a detergent component said region (e.g., of SEQ ID NO: 2 oranother parent xanthan lyase) is relatively more exposed to saiddetergent component than one or more or all of its adjacent regions.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having an alteration(e.g., a substitution, deletion or insertion) at one or more positionsin a region selected from the group of regions 1-6 (e.g., of SEQ ID NO:2 or another parent xanthan lyase), wherein in an aqueous solutioncomprising a detergent component said region (e.g., of SEQ ID NO: 2 oranother parent xanthan lyase) is relatively more conformationallydynamic than one or more or all of its adjacent regions.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having an alteration(e.g., a substitution, deletion or insertion) at one or more positionsin a region selected from the group of regions 1-6 (e.g., of SEQ ID NO:2 or another parent xanthan lyase), wherein in an aqueous solutioncomprising a detergent component said region (e.g., of SEQ ID NO: 2 oranother parent xanthan lyase) is relatively more receptive to deuteriumincorporation than one or more or all of its adjacent regions.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant of the presentdisclosure, comprising an alteration (e.g., a substitution, deletion orinsertion) at one or more positions in two or more regions selected fromthe group of: region 1 corresponding to amino acids 154 to 176 of SEQ IDNO: 2, region 2 corresponding to amino acids 614 to 658 of SEQ ID NO: 2,region 3 corresponding to amino acids 731 to 803 of SEQ ID NO: 2, region4 corresponding to amino acids 807 to 846 of SEQ ID NO: 2, region 5corresponding to amino acids 872 to 885 of SEQ ID NO: 2, and region 6corresponding to amino acids 903 to 1004 of SEQ ID NO: 2, wherein saidvariant has at least about 60% and less than about 100% sequenceidentity to SEQ ID NO: 2, preferably said variant has activity onxanthan gum, further preferably said activity is a xanthan gum degradingactivity.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having multiplealterations (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) in one region (e.g.,of SEQ ID NO: 2 or another parent xanthan lyase) selected from the groupof: region 1 corresponding to amino acids 154 to 176 of SEQ ID NO: 2,region 2 corresponding to amino acids 614 to 658 of SEQ ID NO: 2, region3 corresponding to amino acids 731 to 803 of SEQ ID NO: 2, region 4corresponding to amino acids 807 to 846 of SEQ ID NO: 2, region 5corresponding to amino acids 872 to 885 of SEQ ID NO: 2, and region 6corresponding to amino acids 903 to 1004 of SEQ ID NO: 2, wherein saidvariant has at least about 60% and less than about 100% sequenceidentity to SEQ ID NO: 2, preferably said variant has activity onxanthan gum, further preferably said activity is a xanthan gum degradingactivity.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant having multiplealterations (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) in multiple regions(e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) (e.g., of SEQ ID NO: 2 or anotherparent xanthan lyase) selected from the group of: region 1 correspondingto amino acids 154 to 176 of SEQ ID NO: 2, region 2 corresponding toamino acids 614 to 658 of SEQ ID NO: 2, region 3 corresponding to aminoacids 731 to 803 of SEQ ID NO: 2, region 4 corresponding to amino acids807 to 846 of SEQ ID NO: 2, region 5 corresponding to amino acids 872 to885 of SEQ ID NO: 2, and region 6 corresponding to amino acids 903 to1004 of SEQ ID NO: 2, wherein said variant has at least about 60% andless than about 100% sequence identity to SEQ ID NO: 2, preferably saidvariant has activity on xanthan gum, further preferably said activity isa xanthan gum degrading activity.

In one embodiment the present disclosure relates to a detergentcomposition comprising xanthan lyase variants, comprising an alteration(e.g., a substitution, deletion or insertion) at one or more positionsof the mature parent polypeptide (e.g., SEQ ID NO: 2), wherein eachalteration is independently a substitution, insertion or deletion,wherein the variant has xanthan lyase activity.

In an embodiment, the variant has sequence identity of at least about60%, e.g., at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99%, but less than about 100%, to the aminoacid sequence of the parent xanthan lyase.

In one embodiment, the variant has at least about 60%, e.g., at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,such as at least about 96%, at least about 97%, at least about 98%, orat least about 99%, but less than about 100%, sequence identity to themature polypeptide of SEQ ID NO: 2.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant as described herein,having at least about 61%, at least about 62%, at least about 63%, atleast about 64%, at least about 65%, at least about 66%, at least about67%, at least about 68%, at least about 69%, at least about 70%, atleast about 71%, at least about 72%, at least about 73%, at least about74%, at least about 75%, at least about 76%, at least about 77%, atleast about 78%, at least about 79%, at least about 80%, at least about81%, at least about 82%, at least about 83%, at least about 84%, atleast about 85%, at least about 86%, at least about 87%, at least about88%, at least about 89%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% sequence identity to SEQ ID NO: 2.

In another aspect, a variant comprises an alteration at one or morepositions corresponding to positions 155, 159, 620, 624, 626, 631, 635,645, 649, 650, 656, 738, 745, 746, 748, 752, 753, 754, 757, 764, 769,774, 775, 777, 779, 782, 785, 786, 789, 792, 796, 799, 800, 801, 819,824, 843, 845, 875, 903, 911, 912, 915, 919, 921, 923, 925, 927, 928,930, 932, 933, 941, 966, 967, 991 and 998. In another aspect, a variantcomprises an alteration at two positions corresponding to any ofpositions 155, 159, 620, 624, 626, 631, 635, 645, 649, 650, 656, 738,745, 746, 748, 752, 753, 754, 757, 764, 769, 774, 775, 777, 779, 782,785, 786, 789, 792, 796, 799, 800, 801, 819, 824, 843, 845, 875, 903,911, 912, 915, 919, 921, 923, 925, 927, 928, 930, 932, 933, 941, 966,967, 991 and 998. In another aspect, a variant comprises an alterationat three positions corresponding to any of positions 155, 159, 620, 624,626, 631, 635, 645, 649, 650, 656, 738, 745, 746, 748, 752, 753, 754,757, 764, 769, 774, 775, 777, 779, 782, 785, 786, 789, 792, 796, 799,800, 801, 819, 824, 843, 845, 875, 903, 911, 912, 915, 919, 921, 923,925, 927, 928, 930, 932, 933, 941, 966, 967, 991 and 998. In anotheraspect, a variant comprises an alteration at four or more positions,e.g. five, six, seven, eight, nine, ten or more positions, correspondingto positions 155, 159, 620, 624, 626, 631, 635, 645, 649, 650, 656, 738,745, 746, 748, 752, 753, 754, 757, 764, 769, 774, 775, 777, 779, 782,785, 786, 789, 792, 796, 799, 800, 801, 819, 824, 843, 845, 875, 903,911, 912, 915, 919, 921, 923, 925, 927, 928, 930, 932, 933, 941, 966,967, 991 and 998.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 155. In another aspect, the aminoacid at a position corresponding to position 155 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution Y155E of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 159. In another aspect, the aminoacid at a position corresponding to position 159 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A159P.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 620. In another aspect, the aminoacid at a position corresponding to position 620 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K620R of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 624. In another aspect, the aminoacid at a position corresponding to position 624 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A624E of the maturepolypeptide of SEQ ID NO: 2

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 626. In another aspect, the aminoacid at a position corresponding to position 626 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A626Q of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 631. In another aspect, the aminoacid at a position corresponding to position 631 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T631N or T631E of the maturepolypeptide of SEQ ID NO: 2. A preferred substitution at a positioncorresponding to position 631 is T631N.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 635. In another aspect, the aminoacid at a position corresponding to position 635 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution S635E, S635T or S635Q. Apreferred substitution at a position corresponding to position 635 isS635E.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 649. In another aspect, the aminoacid at a position corresponding to position 649 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T649V, T649K or T649R of themature polypeptide of SEQ ID NO: 2. A preferred substitution at aposition corresponding to position 649 is T649K.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 650. In another aspect, the aminoacid at a position corresponding to position 650 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution Q650G of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 656. In another aspect, the aminoacid at a position corresponding to position 656 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution I656V of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 738. In another aspect, the aminoacid at a position corresponding to position 738 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution G738L of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 745. In another aspect, the aminoacid at a position corresponding to position 745 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K745R of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 746. In another aspect, the aminoacid at a position corresponding to position 746 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution F746L of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 748. In another aspect, the aminoacid at a position corresponding to position 748 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution L748T of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 752. In another aspect, the aminoacid at a position corresponding to position 752 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution P752R or P752K of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 753. In another aspect, the aminoacid at a position corresponding to position 753 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution G753E, G753Q or G753S of themature polypeptide of SEQ ID NO: 2. A preferred substitution at aposition corresponding to position 753 is G753E.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 754. In another aspect, the aminoacid at a position corresponding to position 754 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution S754E, S754L, S754Q or S754Rof the mature polypeptide of SEQ ID NO: 2. A preferred substitution at aposition corresponding to position 754 is S754E or S754R.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 757. In another aspect, the aminoacid at a position corresponding to position 757 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution S757D, S757P or S757E of themature polypeptide of SEQ ID NO: 2. A preferred substitution at aposition corresponding to position 757 is S757D.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 764. In another aspect, the aminoacid at a position corresponding to position 764 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution P764V or P764K of the maturepolypeptide of SEQ ID NO: 2. A preferred substitution at a positioncorresponding to position 764 is P764V.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 769. In another aspect, the aminoacid at a position corresponding to position 769 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the alteration A769D, A769T, A769R, A769S,A769E, A769Q or A769* of the mature polypeptide of SEQ ID NO: 2. Apreferred substitution at a position corresponding to position 769 isA769D.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 774. In another aspect, the aminoacid at a position corresponding to position 774 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A774V of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 775. In another aspect, the aminoacid at a position corresponding to position 775 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution L775A or L775F or L775I orL775M or L775Q or L775S or L775Y of the mature polypeptide of SEQ ID NO:2. A preferred substitution at a position corresponding to position 775is L775M, L775Y or L775A.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 779. In another aspect, the aminoacid at a position corresponding to position 779 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution P779V of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 782. In another aspect, the aminoacid at a position corresponding to position 782 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution Y782I.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 786. In another aspect, the aminoacid at a position corresponding to position 786 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution N786K.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 789. In another aspect, the aminoacid at a position corresponding to position 789 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution G789R.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 792. In another aspect, the aminoacid at a position corresponding to position 792 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K792W, K792Y, K792V or K792Aof the mature polypeptide of SEQ ID NO: 2. A preferred substitution at aposition corresponding to position 792 is K792W or K792Y.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 796. In another aspect, the aminoacid at a position corresponding to position 796 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution N796Q of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 799. In another aspect, the aminoacid at a position corresponding to position 799 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A799H of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 800. In another aspect, the aminoacid at a position corresponding to position 800 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution V800P of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 801. In another aspect, the aminoacid at a position corresponding to position 801 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution D801G of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 819. In another aspect, the aminoacid at a position corresponding to position 819 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K819R or K819T of the maturepolypeptide of SEQ ID NO: 2. A preferred substitution at a positioncorresponding to position 819 is K819R or K819T.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 824. In another aspect, the aminoacid at a position corresponding to position 824 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K824R of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 843. In another aspect, the aminoacid at a position corresponding to position 843 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A843P.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 845. In another aspect, the aminoacid at a position corresponding to position 845 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. A preferred substitution at a positioncorresponding to position 845 is D845E.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 875. In another aspect, the aminoacid at a position corresponding to position 875 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K875T or K875E of the maturepolypeptide of SEQ ID NO: 2. A preferred substitution at a positioncorresponding to position 875 is K875T.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 903. In another aspect, the aminoacid at a position corresponding to position 903 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T903A or T903Q of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 911. In another aspect, the aminoacid at a position corresponding to position 911 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A911M, A911V or A911S of themature polypeptide of SEQ ID NO: 2. A preferred substitution at aposition corresponding to position 911 is A911V.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 912. In another aspect, the aminoacid at a position corresponding to position 912 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A912I or A912T or A912Y of themature polypeptide of SEQ ID NO: 2. A preferred substitution at aposition corresponding to position 912 is A912T.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 915. In another aspect, the aminoacid at a position corresponding to position 915 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T915S, T915Q, T915A or T915Vof the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 919. In another aspect, the aminoacid at a position corresponding to position 919 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T919D, T919F or T919G of themature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 921. In another aspect, the aminoacid at a position corresponding to position 921 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T921R or T921S of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 923. In another aspect, the aminoacid at a position corresponding to position 923 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T923D or T923H of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 925. In another aspect, the aminoacid at a position corresponding to position 925 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T925D or T925Q or T925R of themature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 927. In another aspect, the aminoacid at a position corresponding to position 927 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T927K of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 928. In another aspect, the aminoacid at a position corresponding to position 928 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution D928W of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 930. In another aspect, the aminoacid at a position corresponding to position 930 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution Y930F or Y930H or Y930L of themature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 933. In another aspect, the aminoacid at a position corresponding to position 933 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution D933M of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 941. In another aspect, the aminoacid at a position corresponding to position 941 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution G941D or G941E of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 966. In another aspect, the aminoacid at a position corresponding to position 966 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A966P of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 991. In another aspect, the aminoacid at a position corresponding to position 991 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution N991D of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 998. In another aspect, the aminoacid at a position corresponding to position 998 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution V998K of the maturepolypeptide of SEQ ID NO: 2.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant, having an alteration atone or more positions selected from the group of positions: 155, 159,620, 624, 626, 631, 635, 645, 649, 650, 656, 738, 745, 746, 748, 752,753, 754, 757, 764, 769, 774, 775, 777, 779, 782, 785, 786, 789, 792,796, 799, 800, 801, 819, 824, 843, 845, 875, 903, 911, 912, 915, 919,921, 923, 925, 927, 928, 930, 932, 933, 941, 966, 967, 991 and 998 ofSEQ ID NO: 2, wherein each position corresponds to the positions of SEQID NO: 2.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant, having an alteration atone or more positions selected from the group of: Y155E, A159P, K620R,A624E, A626G, T631N, T631E, S635E, S635T, S635Q, A645S, T649V, T649K,T649R, Q650G, I656V, G738L, K745R, F746L, L748T, P752R, P752K, G753E,G753Q, G753S, S754E, S754L, S754Q, S754R, S757D, S757P, S757E, P764V,P764K, A769D, A769T, A769R, A769S, A769E, A769Q, A769*, A774V, L775M,L775Y, L775A, L775I, L775S, L775F, L775Q, D777K, D777R, P779V, Y782I,A785T, N786K, G789R, K792W, K792Y, K792V, K792A, N796Q, A799H, V800P,D801G, K819R, K819T, K824R, A843P, D845E, K875T, K875E, T903A, T903Q,A911V, A911M, A911S, A912T, A912I, A912Y, T915Q, T915S, T915V, T915A,T919F, T919G, T919D, T921R, T921S, T923H, T923D, T925Q, T925D, T925R,T927K, D928W, Y930H, Y930L, Y930F, A932P, D933M, G941E, G941D, A966P,A967D, N991D and V998K, wherein numbering is according to SEQ ID NO: 2.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 1 herein.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 2 herein.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 3 herein.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 4 herein.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 5 herein.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 6 herein.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 7 herein.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 8 herein.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 9 herein.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 10 herein.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 11 herein.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 12 herein.

In a particular embodiment, the present disclosure relates to adetergent composition comprising a xanthan lyase variant selected fromthe group of the xanthan lyase variants set forth in Table 13 herein.

The variants may further comprise one or more additional alterations atone or more other positions.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof from about 1 to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to from about 20 to about 25residues; or a small extension that facilitates purification by changingnet charge or another function, such as a poly-histidine tract, anantigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for xanthan lyase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J Mol. Biol. 224: 899-904; Wlodaver et al.,1992, FEBS Lett 309: 59-64. The identity of essential amino acids canalso be inferred from an alignment with a related polypeptide.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant, having a total number ofalterations compared to SEQ ID NO: 2 between 1 and about 20, e.g.,between 1 and about 10 or between 1 and about 5, such as about 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 alterations.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant, having an activity onxanthan gum, preferably said activity on xanthan gum is a xanthan gumdegrading activity, further preferably said xanthan gum degradingactivity is EC 4.2.2.12 activity.

In an embodiment, the variant has an improved stability in a detergentcomposition compared to a parent enzyme (e.g., SEQ ID NO: 2).

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant, having an improvedstability in a detergent composition compared to the parent xanthanlyase (e.g., with SEQ ID NO: 2); preferably said detergent composition(in which it has an improved stability and optionally also the detergentcomposition as contemplated herein) comprises a chelator; furtherpreferably said chelator is EDTA or citrate.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant, having a half-lifeimprovement factor (HIF) of >1.0; preferably having a half-lifeimprovement factor (HIF) of > about 1.0, relative to a parent xanthanlyase. More preferably, the half-life improvement factor (HIF) of avariant of the present disclosure is at least about 1.2, such as atleast about 1.5, e.g. at least about 2.0, at least about 3.0, at leastabout 4.0 or at least about 5.0. A preferred way of calculating ahalf-life improvement factor (HIF) is described in example 4 herein.

In one embodiment the present disclosure relates to a detergentcomposition comprising a xanthan lyase variant, wherein a half-lifeimprovement factor (HIF) is determined after incubation of said xanthanlyase variant in a detergent composition at about 25° C. or about 30° C.for a time period from about 30 min to about 20 hours.

Parent

The parent xanthan lyase may be (a) a polypeptide having at least about60% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) apolypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, or (ii) the full-length complement of (i); or (c) apolypeptide encoded by a polynucleotide having at least 60% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1.

In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 2 of at least about 60%, e.g., at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or about 100%, which have xanthan lyase activity. In one aspect,the amino acid sequence of the parent differs by up to about 10 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 2. In another aspect,the parent is a fragment of the mature polypeptide of SEQ ID NO: 2containing at least about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94% or about 95%of the number of amino acids of SEQ ID NO: 2. In another embodiment, theparent is an allelic variant of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, or (ii) the full-length complement of (i) (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,New York).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encoding a parentfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least about 25, at least about 35, or at least about 70nucleotides in length. Preferably, the nucleic acid probe is at leastabout 100 nucleotides in length, e.g., at least about 200 nucleotides,at least about 300 nucleotides, at least about 400 nucleotides, at leastabout 500 nucleotides, at least about 600 nucleotides, at least about700 nucleotides, at least about 800 nucleotides, or at least about 900nucleotides in length. Both DNA and RNA probes can be used. The probesare typically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present disclosure.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present disclosure, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) the full-length complement thereof; or (iv) asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection methods known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe is apolynucleotide that encodes the polypeptide of SEQ ID NO: 2; the maturepolypeptide thereof or a fragment thereof. In another aspect, thenucleic acid probe is SEQ ID NO: 1.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least about 60%, e.g., at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or about 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present disclosure. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present disclosure. Techniquesfor producing fusion polypeptides are known in the art, and includeligating the coding sequences encoding the polypeptides so that they arein frame and that expression of the fusion polypeptide is under controlof the same promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present disclosure, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial enzyme. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, or Streptomyces enzyme, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma enzyme.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulars, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis enzyme.

In another aspect, the parent is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus enzyme.

In another aspect, the parent is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans enzyme.

The parent may be a fungal enzyme. For example, the parent may be ayeast enzyme such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia enzyme; or a filamentous fungal enzymesuch as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria enzyme.

In another aspect, the parent is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis enzyme.

In another aspect, the parent is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia sub thermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride enzyme.

In another aspect, the parent is a Paenibacillus sp. xanthan lyase,e.g., the xanthan lyase of SEQ ID NO: 2.

It will be understood that for the aforementioned species, the presentdisclosure encompasses both the perfect and imperfect states, and othertaxonomic equivalents, e.g., anamorphs, regardless of the species nameby which they are known. Those skilled in the art will readily recognizethe identity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).Preparation of variants

Methods for obtaining a variant having xanthan lyase activity, maycomprise: (a) introducing into a parent xanthan lyase an alteration atone or more positions corresponding to positions 155, 159, 620, 624,626, 631, 635, 645, 649, 650, 656, 738, 745, 746, 748, 752, 753, 754,757, 764, 769, 774, 775, 777, 779, 782, 785, 786, 789, 792, 796, 799,800, 801, 819, 824, 843, 845, 875, 903, 911, 912, 915, 919, 921, 923,925, 927, 928, 930, 932, 933, 941, 966, 967, 991 and 998 of the maturepolypeptide of SEQ ID NO: 2, wherein the variant has xanthan lyaseactivity; and (b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more mutationsare introduced at one or more defined sites in a polynucleotide encodingthe parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentdisclosure. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Embodiments

In one embodiment the present disclosure relates to a detergentcomposition comprising at least one (e.g. about: 1, 2, 3, 4, 5, 6, 7, 8,9 or 10) xanthan lyase variant as disclosed herein.

In one embodiment the present disclosure relates to a detergentcomposition comprising at least one (e.g. about: 1, 2, 3, 4, 5, 6, 7, 8,9 or 10) xanthan lyase variant as disclosed herein, further comprisingone or more detergent components; preferably said detergent componentcomprises a chelator; further preferably said chelator is EDTA orcitrate.

In one embodiment the present disclosure relates to a detergentcomposition comprising at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or10) xanthan lyase variant, further comprising one or more additionalenzymes selected from the group comprising or consisting of:endoglucanases, proteases, amylases, lipases, cutinases, cellulases,xanthan lyases, xyloglucanases, pectinases, pectin lyases, xanthanases,peroxidases, haloperoxygenases, catalases and mannanases, or any mixturethereof.

In one embodiment the present disclosure relates to a detergentcomposition comprising at least one (e.g. about: 1, 2, 3, 4, 5, 6, 7, 8,9 or 10) xanthan lyase variant, wherein said composition furthercomprises one or more detergent components and one or more additionalenzymes selected from the group comprising or consisting of:endoglucanases, proteases, amylases, lipases, cutinases, cellulases,xanthan lyases, xyloglucanases, pectinases, pectin lyases, xanthanases,peroxidases, haloperoxygenases, catalases and mannanases, or any mixturethereof, preferably said detergent component comprises a chelator;further preferably said chelator is EDTA or citrate.

In one embodiment the present disclosure relates to a detergentcomposition comprising at least one (e.g. about: 1, 2, 3, 4, 5, 6, 7, 8,9 or 10) xanthan lyase variant, wherein said composition furthercomprises one or more detergent components, wherein said detergentcomposition is in form of a bar, a homogenous tablet, a tablet havingtwo or more layers, a pouch having one or more compartments, a regularor compact powder, a granule, a paste, a gel, or a regular, compact orconcentrated liquid.

In one embodiment the present disclosure relates to use of a detergentcomposition of the present disclosure, wherein said use is selected fromthe group comprising or consisting of: use for degrading xanthan gum anduse in a cleaning process, such as laundry or hard surface cleaning suchas dish wash.

In one embodiment the present disclosure relates to use of a detergentcomposition of the present disclosure, wherein said detergentcomposition has an enzyme detergency benefit

In one embodiment the present disclosure relates to a method fordegrading xanthan gum comprising: applying a detergent composition ofthe present disclosure to a xanthan gum.

In one embodiment the present disclosure relates to a method fordegrading xanthan gum comprising: applying a detergent composition ofthe present disclosure to a xanthan gum, wherein said xanthan gum is onthe surface of a textile or hard surface, such as dish wash.

Polynucleotides

Disclosed herein are isolated polynucleotides encoding a variant of thepresent disclosure. Said isolated polynucleotides encoding a variant maycomprise: (a) a polynucleotide encoding a variant comprising analteration at one or more positions in at least one chelator-inducedinstability region selected from the group of: i) region 1 correspondingto amino acids 154 to 176 of SEQ ID NO: 2, ii) region 2 corresponding toamino acids 614 to 658 of SEQ ID NO: 2, iii) region 3 corresponding toamino acids 731 to 803 of SEQ ID NO: 2, iv) region 4 corresponding toamino acids 807 to 846 of SEQ ID NO: 2, v) region 5 corresponding toamino acids 872 to 885 of SEQ ID NO: 2, and vi) region 6 correspondingto amino acids 903 to 1004 of SEQ ID NO: 2.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well-known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned in a strain of Bacillus subtilis or E.coli, or a related organism and thus, for example, may be an allelic orspecies variant of the polypeptide encoding region of thepolynucleotide.

Modification of a polynucleotide encoding a polypeptide as describedherein may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1, e.g., asubsequence thereof, and/or by introduction of nucleotide substitutionsthat do not result in a change in the amino acid sequence of thepolypeptide, but which correspond to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions that may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,Ford et al., (1991), ‘Protein Expression and Purification’, 2: 95-107.

Nucleic Acid Constructs

Also disclosed herein are nucleic acid constructs comprising apolynucleotide encoding a variant as described herein operably linked toone or more control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Accordingly, disclosed are nucleic acid constructscomprising a polynucleotide encoding a variant, comprising: (a) apolynucleotide encoding a variant comprising an alteration at one ormore positions in at least one chelator-induced instability regionselected from the group of: i) region 1 corresponding to amino acids 154to 176 of SEQ ID NO: 2, ii) region 2 corresponding to amino acids 614 to658 of SEQ ID NO: 2, iii) region 3 corresponding to amino acids 731 to803 of SEQ ID NO: 2, iv) region 4 corresponding to amino acids 807 to846 of SEQ ID NO: 2, v) region 5 corresponding to amino acids 872 to 885of SEQ ID NO: 2, and vi) region 6 corresponding to amino acids 903 to1004 of SEQ ID NO: 2, operably linked to one or more control sequencesthat direct the expression of the coding sequence in a suitable hostcell under conditions compatible with the control sequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present disclosure. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present disclosure in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present disclosure in a filamentousfungal host cell are promoters obtained from the genes for Aspergillusnidulans acetamidase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKAamylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Fusarium oxysporum trypsin-like protease (WO96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusariumvenenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900),Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase,Trichoderma reesei beta-glucosidase, Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,Trichoderma reesei xanthan lyase I, Trichoderma reesei xanthan lyase II,Trichoderma reesei xanthan lyase III, Trichoderma reesei xanthan lyaseIV, Trichoderma reesei xanthan lyase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, aswell as the NA2-tpi promoter (a modified promoter from an Aspergillusneutral alpha-amylase gene in which the untranslated leader has beenreplaced by an untranslated leader from an Aspergillus triose phosphateisomerase gene; non-limiting examples include modified promoters from anAspergillus niger neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillusnidulans or Aspergillus oryzae triose phosphate isomerase gene); andmutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present disclosure.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cells areobtained from the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens xanthan lyase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

Also disclosed herein are recombinant expression vectors comprising apolynucleotide encoding a variant as described herein, a promoter, andtranscriptional and translational stop signals. Accordingly, alsodisclosed are methods for obtaining recombinant expression vectorscomprising a polynucleotide encoding a variant, comprising: (a) apolynucleotide encoding a variant comprising an alteration at one ormore positions in at least one chelator-induced instability regionselected from the group of: i) region 1 corresponding to amino acids 154to 176 of SEQ ID NO: 2, ii) region 2 corresponding to amino acids 614 to658 of SEQ ID NO: 2, iii) region 3 corresponding to amino acids 731 to803 of SEQ ID NO: 2, iv) region 4 corresponding to amino acids 807 to846 of SEQ ID NO: 2, v) region 5 corresponding to amino acids 872 to 885of SEQ ID NO: 2, and vi) region 6 corresponding to amino acids 903 to1004 of SEQ ID NO: 2, a promoter, and transcriptional and translationalstop signals.

The various nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the polypeptide at such sites.Alternatively, the polynucleotide may be expressed by inserting thepolynucleotide or a nucleic acid construct comprising the polynucleotideinto an appropriate vector for expression. In creating the expressionvector, the coding sequence is located in the vector so that the codingsequence is operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any mechanism for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as from about 100 to about 10,000 basepairs, from about 400 to about 10,000 base pairs, and from about 800 toabout 10,000 base pairs, which have a high degree of sequence identityto the corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell. Furthermore, the integrational elements may be non-encoding orencoding polynucleotides. On the other hand, the vector may beintegrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present disclosure may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present disclosure are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

Also disclosed herein are recombinant host cells, comprising apolynucleotide encoding a variant as described herein operably linked toone or more control sequences that direct the production of a variant ofthe present disclosure. Accordingly, disclosed herein are recombinanthost cells, comprising a polynucleotide encoding a variant as describedherein, comprising: (a) a polynucleotide encoding a variant comprisingan alteration at one or more positions in at least one chelator-inducedinstability region selected from the group of: i) region 1 correspondingto amino acids 154 to 176 of SEQ ID NO: 2, ii) region 2 corresponding toamino acids 614 to 658 of SEQ ID NO: 2, iii) region 3 corresponding toamino acids 731 to 803 of SEQ ID NO: 2, iv) region 4 corresponding toamino acids 807 to 846 of SEQ ID NO: 2, v) region 5 corresponding toamino acids 872 to 885 of SEQ ID NO: 2, and vi) region 6 correspondingto amino acids 903 to 1004 of SEQ ID NO: 2, operably linked to one ormore control sequences that direct the production of a variant.

A construct or vector comprising a polynucleotide is introduced into ahost cell so that the construct or vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector as describedearlier. The term “host cell” encompasses any progeny of a parent cellthat is not identical to the parent cell due to mutations that occurduring replication. The choice of a host cell will to a large extentdepend upon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present disclosure, e.g., a prokaryote or aeukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, 1 Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this present disclosure, yeast shall be defined as describedin Biology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally exemplified by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

Also disclosed herein are methods of producing (e.g., in vitro or exvivo methods) a variant as described herein, comprising: (a) cultivatinga host cell of the present disclosure under conditions suitable forexpression of the variant; and (b) recovering the variant. Accordingly,disclosed herein are methods of producing (e.g., in vitro or ex vivomethods) a variant, comprising: (a) cultivating a host cell comprising apolynucleotide encoding a variant comprising an alteration at one ormore positions in at least one chelator-induced instability regionselected from the group of: i) region 1 corresponding to amino acids 154to 176 of SEQ ID NO: 2, ii) region 2 corresponding to amino acids 614 to658 of SEQ ID NO: 2, iii) region 3 corresponding to amino acids 731 to803 of SEQ ID NO: 2, iv) region 4 corresponding to amino acids 807 to846 of SEQ ID NO: 2, v) region 5 corresponding to amino acids 872 to 885of SEQ ID NO: 2, and vi) region 6 corresponding to amino acids 903 to1004 of SEQ ID NO: 2, under conditions suitable for expression of thevariant, and (b) optionally, recovering the variant.

Further disclosed are methods of producing (e.g., in vitro or ex vivomethods) a variant of the present disclosure, comprising (a) cultivatinga cell, which in its wild-type form produces the polypeptide, underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide. In a preferred aspect, the cell is aPaenibacillus cell, or a Microbacterium cell.

Also disclosed are methods of producing (e.g., in vitro or ex vivomethods) a variant of the present disclosure, comprising (a) cultivatinga recombinant host cell of the present disclosure under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The variant polypeptide may be detected using methods known in the artthat are specific for the polypeptides such as methods for determiningcellulose or xanthan lyase activity. These detection methods include,but are not limited to, use of specific antibodies, formation of anenzyme product, or disappearance of an enzyme substrate. For example, anenzyme assay may be used to determine the activity of the polypeptide.

The variant polypeptide may be recovered using methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant polypeptide may be purified by a variety of procedures knownin the art including, but not limited to, chromatography (e.g., ionexchange, affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the variant polypeptide is not recovered, butrather a host cell expressing the polypeptide is used as a source of thevariant polypeptide.

Compositions

In one certain aspect, the variants as contemplated herein have improvedstability in detergents compared to a parent enzyme or compared to axanthan lyase having the identical amino acid sequence of the variant,but not having an alteration (e.g., a substitution, deletion orinsertion) at one or more of the specified positions or compared to thexanthan lyase with SEQ ID NO: 2, wherein activity and/or stability indetergent is measured as disclosed in example 4 herein.

Besides enzymes the detergent compositions may comprise additionalcomponents. The choice of additional components is within the skill ofthe artisan and includes conventional ingredients, including theexemplary non-limiting components set forth below. The choice ofcomponents may include, for fabric care, the consideration of the typeof fabric to be cleaned, the type and/or degree of soiling, thetemperature at which cleaning is to take place, and the formulation ofthe detergent product. Although components mentioned below arecategorized by general header according to a particular functionality,this is not to be construed as a limitation, as a component may compriseadditional functionalities as will be appreciated by the skilledartisan.

The detergent composition may be suitable for the laundering of textilessuch as e.g. fabrics, cloths or linen, or for cleaning hard surfacessuch as e.g. floors, tables, or dish wash.

Detergent Compositions

In one embodiment, a variantas described herein may be added to adetergent composition in an amount corresponding to from about 0.0001 toabout 200 mg of enzyme protein, such as from about 0.0005 to about 100mg of enzyme protein, preferably from about 0.001 to about 30 mg ofenzyme protein, more preferably from about 0.005 to about 8 mg of enzymeprotein, even more preferably from about 0.01 to about 2 mg of enzymeprotein per litre of wash liquor.

A composition for use in automatic dishwash (ADW), for example, mayinclude from about 0.0001% to about 50%, such as from about 0.001% toabout 20%, such as from about 0.01% to about 10%, such as from about0.05 to about 5% of enzyme protein by weight of the composition.

A composition for use in laundry granulation or a solid/granular laundrycomposition in general, for example, may include from about 0.0001% toabout 50%, such as from about 0.001% to about 20%, such as from about0.01% to about 10%, such as from about 0.05% to about 5% of enzymeprotein by weight of the composition.

A composition for use in laundry liquid, for example, may include fromabout 0.0001% to about 10%, such as from about 0.001 to about 7%, suchas from about 0.1% to about 5% of enzyme protein by weight of thecomposition.

The enzyme(s) of the detergent composition of the present disclosure maybe stabilized using conventional stabilizing agents, e.g., a polyol suchas propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example,WO92/19709 and WO92/19708.

In certain markets different wash conditions and, as such, differenttypes of detergents are used. This is disclosed in e.g. EP 1 025 240.For example, In Asia (Japan) a low detergent concentration system isused, while the United States uses a medium detergent concentrationsystem, and Europe uses a high detergent concentration system.

A low detergent concentration system includes detergents where less thanabout 800 ppm of detergent components are present in the wash water.Japanese detergents are typically considered low detergent concentrationsystem as they have approximately 667 ppm of detergent componentspresent in the wash water.

A medium detergent concentration includes detergents where between about800 ppm and about 2000 ppm of detergent components are present in thewash water. North American detergents are generally considered to bemedium detergent concentration systems as they have approximately 975ppm of detergent components present in the wash water.

A high detergent concentration system includes detergents where greaterthan about 2000 ppm of detergent components are present in the washwater. European detergents are generally considered to be high detergentconcentration systems as they have approximately 4500-5000 ppm ofdetergent components in the wash water.

Latin American detergents are generally high suds phosphate builderdetergents and the range of detergents used in Latin America can fall inboth the medium and high detergent concentrations as they range fromabout 1500 ppm to about 6000 ppm of detergent components in the washwater. Such detergent compositions are all embodiments of the presentdisclosure.

A polypeptide of the present disclosure may also be incorporated in thedetergent formulations disclosed in WO97/07202, which is herebyincorporated by reference.

Surfactants

The detergent composition may comprise one or more surfactants, whichmay be anionic and/or cationic and/or non-ionic and/or semi-polar and/orzwitterionic, or a mixture thereof. In a particular embodiment, thedetergent composition includes a mixture of one or more nonionicsurfactants and one or more anionic surfactants. The surfactant(s) istypically present at a level of from about 0.1% to about 60% by weight,such as about 1% to about 40%, or about 3% to about 20%, or about 3% toabout 10%. The surfactant(s) is chosen based on the desired cleaningapplication, and includes any conventional surfactant(s) known in theart. Any surfactant known in the art for use in detergents may beutilized.

When included therein the detergent will usually comprise from about 1%to about 40% by weight, such as from about 5% to about 30%, includingfrom about 5% to about 15%, or from about 20% to about 25% of an anionicsurfactant. Non-limiting examples of anionic surfactants includesulfates and sulfonates, in particular, linear alkylbenzenesulfonates(LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS),phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates,alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonatesand disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate(SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS),alcohol ethersulfates (AES or AEOS or FES, also known as alcoholethoxysulfates or fatty alcohol ether sulfates), secondaryalkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates,sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methylesters (alpha-SFMe or SES) including methyl ester sulfonate (MES),alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid(DTSA), fatty acid derivatives of amino acids, diesters and monoestersof sulfo-succinic acid or soap, and combinations thereof.

When included therein the detergent will usually comprise from about 0%to about 10% by weight of a cationic surfactant. Non-limiting examplesof cationic surfactants include alklydimethylethanolamine quat (ADMEAQ),cetyltrimethylammonium bromide (CTAB), dimethyldistearylammoniumchloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternaryammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, andcombinations thereof.

When included therein the detergent will usually comprise from about0.2% to about 40% by weight of a non-ionic surfactant, for example fromabout 0.5% to about 30%, in particular from about 1% to about 20%, fromabout 3% to about 10%, such as from about 3% to about 5%, or from about8% to about 12%. Non-limiting examples of non-ionic surfactants includealcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylatedfatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such asethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenolethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides(APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fattyacid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides(EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine(glucamides, GA, or fatty acid glucamide, FAGA), as well as productsavailable under the trade names SPAN and TWEEN, and combinationsthereof.

When included therein the detergent will usually comprise from about 0%to about 10% by weight of a semipolar surfactant. Non-limiting examplesof semipolar surfactants include amine oxides (AO) such asalkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide andN-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acidalkanolamides and ethoxylated fatty acid alkanolamides, and combinationsthereof.

When included therein the detergent will usually comprise from about 0%to about 10% by weight of a zwitterionic surfactant. Non-limitingexamples of zwitterionic surfactants include betaine,alkyldimethylbetaine, sulfobetaine, and combinations thereof.

Hydrotropes

A hydrotrope is a compound that solubilises hydrophobic compounds inaqueous solutions (or oppositely, polar substances in anon-polarenvironment). Typically, hydrotropes have both hydrophilic and ahydrophobic character (so-called amphiphilic properties as known fromsurfactants); however the molecular structure of hydrotropes generallydo not favor spontaneous self-aggregation, see e.g. review by Hodgdonand Kaler (2007), Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not display a critical concentration above whichself-aggregation occurs as found for surfactants and lipids formingmiceller, lamellar or other well defined meso-phases. Instead, manyhydrotropes show a continuous-type aggregation process where the sizesof aggregates grow as concentration increases. However, many hydrotropesalter the phase behaviour, stability, and colloidal properties ofsystems containing substances of polar and non-polar character,including mixtures of water, oil, surfactants, and polymers. Hydrotropesare classically used across industries from pharma, personal care, food,to technical applications. Use of hydrotropes in detergent compositionsallow for example more concentrated formulations of surfactants (as inthe process of compacting liquid detergents by removing water) withoutinducing undesired phenomena such as phase separation or high viscosity.

The detergent may comprise from about 0 to about 5% by weight, such asfrom about 0.5 to about 5%, or about 3% to about 5%, of a hydrotrope.Any hydrotrope known in the art for use in detergents may be utilized.Non-limiting examples of hydrotropes include sodium benzene sulfonate,sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodiumcumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcoholsand polyglycolethers, sodium hydroxynaphthoate, sodiumhydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, andcombinations thereof.

Builders and Co-Builders

The detergent composition may comprise from about 0 to about 65% byweight, such as from about 5% to about 45% of a detergent builder orco-builder, or a mixture thereof. In a dish wash deteregent, the levelof builder is typically from about 40 to about 65%, particularly fromabout 50 to about 65%. The builder and/or co-builder may particularly bea chelating agent that forms water-soluble complexes with Ca and Mg. Anybuilder and/or co-builder known in the art for use in laundry detergentsmay be utilized. Non-limiting examples of builders include zeolites,diphosphates (pyrophosphates), triphosphates such as sodium triphosphate(STP or STPP), carbonates such as sodium carbonate, soluble silicatessuch as sodium metasilicate, layered silicates (e.g., SKS-6 fromHoechst), ethanolamines such as 2-aminoethan-1-ol (MEA), diethanolamine(DEA, also known as iminodiethanol), triethanolamine (TEA, also known as2,2′,2″-nitrilotriethanol), and carboxymethyl inulin (CMI), andcombinations thereof.

The detergent composition may also comprise from about 0 to about 20% byweight, such as from about 5% to about 10%, of a detergent co-builder,or a mixture thereof. The detergent composition may include include aco-builder alone, or in combination with a builder, for example azeolite builder. Non-limiting examples of co-builders includehomopolymers of polyacrylates or copolymers thereof, such aspoly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA/PMA).Further non-limiting examples include citrate, chelators such asaminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- oralkenylsuccinic acid. Additional specific examples include2,2′,2″-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid(IDS), ethylenediamine-N,N′-disuccinic acid (EDDS),methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid(GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP),ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA),diethylenetriaminepentakis(methylenephosphonic acid) (DTPMPA or DTMPA),N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoaceticacid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), asparticacid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid(SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL),N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid(MIDA), α-alanine-N, N-diacetic acid (α-ALDA), serine-N, N-diacetic acid(SEDA), isoserine-N, N-diacetic acid (ISDA), phenylalanine-N, N-diaceticacid (PHDA), anthranilic acid-N, N-diacetic acid (ANDA), sulfanilicacid-N, N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA) andsulfomethyl-N, N-diacetic acid (SMDA),N-(2-hydroxyethyl)-ethylidenediamine-N, N′, N′-triacetate (HEDTA),diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonicacid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), andcombinations and salts thereof. Further exemplary builders and/orco-builders are described in, e.g., WO 09/102854, U.S. Pat. No.5,977,053

Bleaching Systems

The detergent may comprise from about 0 to about 50% by weight, such asfrom about 0.1% to about 25%, of a bleaching system. Any bleachingsystem known in the art for use in laundry detergents may be utilized.Suitable bleaching system components include bleaching catalysts,photobleaches, bleach activators, sources of hydrogen peroxide such assodium percarbonate and sodium perborates, preformed peracids andmixtures thereof. Suitable preformed peracids include, but are notlimited to, peroxycarboxylic acids and salts, percarbonic acids andsalts, perimidic acids and salts, peroxymonosulfuric acids and salts,for example, Oxone (R), and mixtures thereof. Non-limiting examples ofbleaching systems include peroxide-based bleaching systems, which maycomprise, for example, an inorganic salt, including alkali metal saltssuch as sodium salts of perborate (usually mono- or tetra-hydrate),percarbonate, persulfate, perphosphate, persilicate salts, incombination with a peracid-forming bleach activator. The term bleachactivator is meant herein as a compound which reacts with peroxygenbleach like hydrogen peroxide to form a peracid. The peracid thus formedconstitutes the activated bleach. Suitable bleach activators to be usedherein include those belonging to the class of esters amides, imides oranhydrides. Suitable examples are tetracetylethylene diamine (TAED),sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS),diperoxy dodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS),4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS),4-(nonanoyloxy)-benzenesulfonate (NOBS), and/or those disclosed inWO98/17767. A particular family of bleach activators of interest wasdisclosed in EP624154 and particularly preferred in that family isacetyl triethyl citrate (ATC). ATC or a short chain triglyceride liketriacetin has the advantage that it is environmental friendly as iteventually degrades into citric acid and alcohol. Furthermore acetyltriethyl citrate and triacetin has a good hydrolytical stability in theproduct upon storage and it is an efficient bleach activator. FinallyATC provides a good building capacity to the laundry additive.Alternatively, the bleaching system may comprise peroxyacids of, forexample, the amide, imide, or sulfone type. The bleaching system mayalso comprise peracids such as 6-(phthalimido)peroxyhexanoic acid (PAP).The bleaching system may also include a bleach catalyst. In someembodiments the bleach component may be an organic catalyst selectedfrom the group of organic catalysts having the following formulae:

(iii) and mixtures thereof wherein each R¹ is independently a branchedalkyl group containing from about 9 to about 24 carbons or linear alkylgroup containing from about 11 to about 24 carbons, preferably each R¹is independently a branched alkyl group containing from about 9 to about18 carbons or linear alkyl group containing from about 11 to about 18carbons, more preferably each R¹ is independently selected from thegroup of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl,n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl,iso-tridecyl and iso-pentadecyl. Other exemplary bleaching systems aredescribed, e.g. in WO2007/087258, WO2007/087244, WO2007/087259 andWO2007/087242. Suitable photobleaches may for example be sulfonated zincphthalocyanine

Polymers

The detergent may comprise from about 0 to about 10% by weight, such asfrom about 0.5 to about 5%, from about 2 to about 5%, from about 0.5 toabout 2% or from about 0.2 to about 1% of a polymer. Any polymer knownin the art for use in detergents may be utilized. The polymer mayfunction as a co-builder as mentioned above, or may provideantiredeposition, fiber protection, soil release, dye transferinhibition, grease cleaning and/or anti-foaming properties. Somepolymers may have more than one of the above-mentioned properties and/ormore than one of the below-mentioned motifs. Exemplary polymers include(carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA),poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethyleneoxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin(CMI), and polycarboxylates such as PAA, PAA/PMA, poly-aspartic acid,and lauryl methacrylate/acrylic acid copolymers, hydrophobicallymodified CMC (HM-CMC) and silicones, copolymers of terephthalic acid andoligomeric glycols, copolymers of poly(ethylene terephthalate) andpoly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole)(PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO) andpolyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymersinclude sulfonated polycarboxylates, polyethylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated.

Fabric Hueing Agents

The detergent compositions of the present disclosure may also includefabric hueing agents such as dyes or pigments, which when formulated indetergent compositions can deposit onto a fabric when said fabric iscontacted with a wash liquor comprising said detergent compositions andthus altering the tint of said fabric through absorption/reflection ofvisible light. Fluorescent whitening agents emit at least some visiblelight. In contrast, fabric hueing agents alter the tint of a surface asthey absorb at least a portion of the visible light spectrum. Suitablefabric hueing agents include dyes and dye-clay conjugates, and may alsoinclude pigments. Suitable dyes include small molecule dyes andpolymeric dyes. Suitable small molecule dyes include small molecule dyesselected from the group of dyes falling into the Colour Index (C.I.)classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue,Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, ormixtures thereof, for example as described in WO2005/03274,WO2005/03275, WO2005/03276 and EP1876226 (hereby incorporated byreference). The detergent composition preferably comprises from about0.00003 wt % to about 0.2 wt %, from about 0.00008 wt % to about 0.05 wt%, or even from about 0.0001 wt % to about 0.04 wt % fabric hueingagent. The composition may comprise from about 0.0001 wt % to about 0.2wt % fabric hueing agent, this may be especially preferred when thecomposition is in the form of a unit dose pouch. Suitable hueing agentsare also disclosed in, e.g. WO 2007/087257 and WO2007/087243.

Additional Enzymes

The detergent additive as well as the detergent composition may compriseone or more [additional] enzymes such as a xanthan lyase, protease,lipase, cutinase, an amylase, lichenase, carbohydrase, cellulase,pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., alaccase, and/or peroxidase.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases:

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Example of cellulases exhibiting endo-beta-1,4-glucanase activity (EC3.2.1.4) are those having described in WO02/099091.

Other examples of cellulases include the family 45 cellulases describedin WO96/29397, and especially variants thereof having substitution,insertion and/or deletion at one or more of the positions correspondingto the following positions in SEQ ID NO: 8 of WO 02/099091: 2, 4, 7, 8,10, 13, 15, 19, 20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43,44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82,84, 86, 88, 90, 91, 93, 95, 95d, 95h, 95j, 97, 100, 101, 102, 103, 113,114, 117, 119, 121, 133, 136, 137, 138, 139, 140a, 141, 143a, 145, 146,147, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c,160e, 160k, 161, 162, 164, 165, 168, 170, 171, 172, 173, 175, 176, 178,181, 183, 184, 185, 186, 188, 191, 192, 195, 196, 200, and/or 20,preferably selected among P19A, G20K, Q44K, N48E, Q119H or Q146 R.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases:

The additional enzyme may be another protease or protease variant. Theprotease may be of animal, vegetable or microbial origin, includingchemically or genetically modified mutants. Microbial origin ispreferred. It may be an alkaline protease, such as a serine protease ora metalloprotease. A serine protease may for example be of the 51family, such as trypsin, or the S8 family such as subtilisin. Ametalloproteases protease may for example be a thermolysin from e.g.family M4, M5, M7 or M8.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases are a subgroup ofproteases exemplified by having a serine in the active site, which formsa covalent adduct with the substrate. The subtilases may be divided into6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, theProteinase K family, the Lantibiotic peptidase family, the Kexin familyand the Pyrolysin family. In one aspect of the present disclosure theprotease may be a subtilase, such as a subtilisin or a variant hereof.Further the subtilases (and the serine proteases) are exemplified byhaving two active site amino acid residues apart from the serine, namelya histidine and an aspartic acid residue.

Examples of subtilisins are those derived from Bacillus such assubtilisin lentus, Bacillus lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309,subtilisin 147 and subtilisin 168 described in WO 89/06279 and proteasePD138 (WO 93/18140). Additional serine protease examples are describedin WO 98/020115, WO 01/44452, WO 01/58275, WO 01/58276, WO 03/006602 andWO 04/099401. An example of a subtilase variants may be those havingmutations in any of the positions: 3, 4, 9, 15, 27, 36, 68, 76, 87, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129,130, 160, 167, 170, 194, 195, 199, 205, 217, 218, 222, 232, 235, 236,245, 248, 252 and 274 using the BPN′ numbering. More preferred thesubtilase variants may comprise the mutations: S3T, V41, S9R, A15T,K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,RS103A, V1041,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A,G160D, Y167A, R170S, A194P, G195E, V199M, V2051, L217D, N218D, M222S,A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN′ numbering). Afurther preferred protease is the alkaline protease from Bacillus lentusDSM 5483, as described for example in WO 95/23221, and variants thereofwhich are described in WO 92/21760, WO 95/23221, EP 1921147 and EP1921148.

Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO 89/06270 and WO94/25583. Examples of useful proteases are the variants described in WO92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially thevariants with substitutions in one or more of the following positions:27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218,222, 224, 235, and 274. Examples of metalloproteases are the neutralmetalloprotease as described in WO 07/044993.

Preferred commercially available protease enzymes include Alcalase™,Coronase™, Duralase™, Durazym™, Esperase™, Everlase™, Kannase™,Liquanase™, Liquanase Ultra™, Ovozyme™, Polarzyme™, Primase™, Relase™,Savinase™ and Savinase Ultra™, (Novozymes A/S), Axapem™ (Gist-BrocasesN.V.), BLAP and BLAP X (Henkel AG & Co. KGaA), Excellase™, FN2™, FN3™,FN4™, Maxaca™, Maxapem™, Maxatase™, Properase™ Purafast™, Purafect™,Purafect OxP™, Purafect Prime™ and Puramax™ (Genencor int.).

Lipases and Cutinases:

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g. from T.lanuginosus (previously named Humicola lanuginosa) as described inEP258068 and EP305216, cutinase from Humicola, e.g. H. insolens(WO96/13580), lipase from strains of Pseudomonas (some of these nowrenamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes(EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 &WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyceslipases (WO10/065455), cutinase from Magnaporthe grisea (WO10/107560),cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipasefrom Thermobifida fusca (WO11/084412), Geobacillus stearothermophiluslipase (WO11/084417), lipase from Bacillus subtilis (WO11/084599), andlipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis(WO12/137147).

Further examples are lipases sometimes referred to as acyltransferasesor perhydrolases, e.g. acyltransferases with homology to Candidaantarctica lipase A (WO10/111143), acyltransferase from Mycobacteriumsmegmatis (WO05/56782), perhydrolases from the CE 7 family (WO09/67279),and variants of the M. smegmatis perhydrolase in particular the S54Vvariant used in the commercial product Gentle Power Bleach from HuntsmanTextile Effects Pte Ltd (WO10/100028).

Other examples are lipase variants such as those described in EP407225,WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381,WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063,WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial lipase products include include Lipolase™, Lipex™;Lipolex™ and Lipoclean™ (Novozymes A/S), Lumafast (originally fromGenencor) and Lipomax (originally from Gist-Brocades).

Amylases

The amylase may be an alpha-amylase, a beta-amylase or a glucoamylaseand may be of bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Amylases include, for example,alpha-amylases obtained from Bacillus, e.g., a special strain ofBacillus licheniformis, described in more detail in GB 1,296,839.

Examples of amylases are those having SEQ ID NO: 3 in WO 95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferredvariants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQID NO: 4 of WO 99/019467, such as variants with substitutions in one ormore of the following positions: 15, 23, 105, 106, 124, 128, 133, 154,156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243,264, 304, 305, 391, 408, and 444 of SEQ ID NO: 3 in WO 95/10603.

Other amylases are variants of SEQ ID NO: 1 of WO 2016/203064 having atleast 75% sequence identity to SEQ ID NO: 1 thereof. Preferred variantsare variants comprising a modification in one or more positionscorresponding to positions 1, 54, 56, 72, 109, 113, 116, 134, 140, 159,167, 169, 172, 173, 174, 181, 182, 183, 184, 189, 194, 195, 206, 255,260, 262, 265, 284, 289, 304, 305, 347, 391, 395, 439, 469, 444, 473,476, or 477 of SEQ ID NO: 1, wherein said alpha-amylase variant has asequence identity of at least about 75% but less than about 100% to SEQID NO: 1.

Further amylases which can be used are amylases having SEQ ID NO: 6 inWO 02/010355 or variants thereof having about 90% sequence identity toSEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having adeletion in positions 181 and 182 and a substitution in position 193.

Other amylase examples are hybrid alpha-amylase comprising residues fromabout 1 to about 33 of the alpha-amylase derived from B.amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residuesfrom about 36 to about 483 of the B. licheniformis alpha-amylase shownin SEQ ID NO: 4 of WO 2006/066594 or variants having about 90% sequenceidentity thereof. Preferred variants of this hybrid alpha-amylase arethose having a substitution, a deletion or an insertion in one of moreof the following positions: G48, T49, G107, H156, A181, N190, M197,1201, A209 and Q264. Most preferred variants of the hybrid alpha-amylasecomprising residues from about 1 to about 33 of the alpha-amylasederived from B. amylohquefaciens shown in SEQ ID NO: 6 of WO 2006/066594and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:

M197T; H156Y+A181T+N190F+A209V+Q264S; orG48+T49+G107+H156+A181+N190+1201+A209+Q264.

Further amylase examples are amylases having SEQ ID NO: 6 in WO99/019467 or variants thereof having about 90% sequence identity to SEQID NO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, 1206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsG182 and H183 or positions H183 and G184. Additional amylases are thosehaving SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 orvariants thereof having about 90% sequence identity to SEQ ID NO: 1, SEQID NO: 2 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO: 7 are those having a substitution, a deletion or aninsertion in one or more of the following positions: 140, 181, 182, 183,184, 195, 206, 212, 243, 260, 269, 304 and 476. More preferred variantsare those having a deletion in positions 182 and 183 or positions 183and 184. Most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2or SEQ ID NO: 7 are those having a deletion in positions 183 and 184 anda substitution in positions 140, 195, 206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having about90% sequence identity to SEQ ID NO: 2 of WO 08/153815 or about 90%sequence identity to SEQ ID NO: 10 in WO 01/66712. Preferred variants ofSEQ ID NO: 10 in WO 01/66712 are those having a substitution, a deletionor an insertion in one of more of the following positions: 176, 177,178, 179, 190, 201, 207, 211 and 264.

Further amylases which can be used are amylases having SEQ ID NO: 2 ofWO 09/061380 or variants thereof having about 90% sequence identity toSEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those having asubstitution, a deletion or an insertion in one of more of the followingpositions: Q87, Q98, 5125, N128, T131, T165, K178, R180, 5181, T182,G183, M201, F202, N225, 5243, N272, N282, Y305, R309, D319, Q320, Q359,K444 and G475. More preferred variants of SEQ ID NO: 2 are those havingthe substitution in one of more of the following positions: Q87E,R,Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R,N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E and G475K and/ordeletion in position R180 and/or 5181. Most preferred amylase variantsof SEQ ID NO: 2 are those having the substitutions:

N128C+K178L+T182G+Y305R+G475K;N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;S125A+N128C+K178L+T182G+Y305R+G475K; or

S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variantoptionally further comprises a substitution at position 243 and/or adeletion at position 180 and/or position 181.

Other examples of amylases are the alpha-amylase having SEQ ID NO: 12 inWO01/66712 or a variant having at least about 90%, such as at leastabout 95%, sequence identity to SEQ ID NO: 12. Preferred amylasevariants are those having a substitution, a deletion or an insertion inone of more of the following positions of SEQ ID NO: 12 in WO01/66712:R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298,N299, K302, 5303, N306, R310, N314; R320, H324, E345, Y396, R400, W439,R444, N445, K446, Q449, R458, N471, N484. Particular preferred amylasesinclude variants having a deletion of D183 and G184 and having thesubstitutions R118K, N195F, R320K and R458K, and a variant additionallyhaving substitutions in one or more position selected from the group:M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, mostpreferred a variant that additionally has substitutions in all thesepositions.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™,Stainzyme™, Stainzyme Plus™, Natalase™ and BAN™ (Novozymes A/S),Rapidase™ and Purastar™ (from Genencor International Inc.).

Peroxidases/Oxidases:

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the present disclosure, i.e., a separate additive or a combinedadditive, can be formulated, for example, as a granulate, liquid,slurry, etc. Preferred detergent additive formulations are granulates,in particular non-dusting granulates, liquids, in particular stabilizedliquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights offrom about 1000 to about 20000; ethoxylated nonylphenols having fromabout 16 to about 50 ethylene oxide units; ethoxylated fatty alcohols inwhich the alcohol contains from about 12 to about 20 carbon atoms and inwhich there are from about 15 to about 80 ethylene oxide units; fattyalcohols; fatty acids; and mono- and di- and triglycerides of fattyacids. Examples of film-forming coating materials suitable forapplication by fluid bed techniques are given in GB 1483591. Liquidenzyme preparations may, for instance, be stabilized by adding a polyolsuch as propylene glycol, a sugar or sugar alcohol, lactic acid or boricacid according to established methods. Protected enzymes may be preparedaccording to the method disclosed in EP 238,216.

Adjunct Materials

Any detergent components known in the art for use in laundry detergentsmay also be utilized. Other optional detergent components includeanti-corrosion agents, anti-shrink agents, anti-soil redepositionagents, anti-wrinkling agents, bactericides, binders, corrosioninhibitors, disintegrants/disintegration agents, dyes, enzymestabilizers (including boric acid, borates, CMC, and/or polyols such aspropylene glycol), fabric conditioners including clays,fillers/processing aids, fluorescent whitening agents/opticalbrighteners, foam boosters, foam (suds) regulators, perfumes,soil-suspending agents, softeners, suds suppressors, tarnish inhibitors,and wicking agents, either alone or in combination. Any ingredient knownin the art for use in laundry detergents may be utilized. The choice ofsuch ingredients is well within the skill of the artisan.

Dispersants:

The detergent compositions of the present disclosure can also containdispersants. In particular powdered detergents may comprise dispersants.Suitable water-soluble organic materials include the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms. Suitable dispersants are for exampledescribed in Powdered Detergents, Surfactant science series volume 71,Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents:

The detergent compositions of the present disclosure may also includeone or more dye transfer inhibiting agents. Suitable polymeric dyetransfer inhibiting agents include, but are not limited to,polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers ofN-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinylimidazoles or mixtures thereof. When present in a subjectcomposition, the dye transfer inhibiting agents may be present at levelsfrom about 0.0001% to about 10%, from about 0.01% to about 5% or evenfrom about 0.1% to about 3% by weight of the composition.

Fluorescent Whitening Agent:

The detergent compositions of the present disclosure will preferablyalso contain additional components that may tint articles being cleaned,such as fluorescent whitening agent or optical brighteners. Wherepresent the brightener is preferably at a level of from about 0.01% toabout 0.5%. Any fluorescent whitening agent suitable for use in alaundry detergent composition may be used in the composition of thepresent disclosure. The most commonly used fluorescent whitening agentsare those belonging to the classes of diaminostilbene-sulphonic acidderivatives, diarylpyrazoline derivatives and bisphenyl-distyrylderivatives. Examples of the diaminostilbene-sulphonic acid derivativetype of fluorescent whitening agents include the sodium salts of:4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate; 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2′-disulphonate;4,4′-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2′-disulphonate;4,4′-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate and2-(stilbyl-4″-naptho-1,2′:4,5)-1,2,3-trizole-2″-sulphonate. Preferredfluorescent whitening agents are Tinopal DMS and Tinopal CBS availablefrom Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium saltof 4,4′-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbenedisulphonate. Tinopal CBS is the disodium salt of2,2′-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescentwhitening agents is the commercially available Parawhite KX, supplied byParamount Minerals and Chemicals, Mumbai, India. Other fluorescerssuitable for use in the present disclosure include the 1-3-diarylpyrazolines and the 7-alkylaminocoumarins. Suitable fluorescentbrightener levels include lower levels of from about 0.01, from about0.05, from about 0.1 or even from about 0.2 wt % to upper levels of fromabout 0.5 or even from about 0.75 wt %.

Soil Release Polymers:

The detergent compositions of the present disclosure may also includeone or more soil release polymers which aid the removal of soils fromfabrics such as cotton and polyester based fabrics, in particular theremoval of hydrophobic soils from polyester based fabrics. The soilrelease polymers may for example be nonionic or anionic terephthaltebased polymers, polyvinyl caprolactam and related copolymers, vinylgraft copolymers, polyester polyamides see for example Chapter 7 inPowdered Detergents, Surfactant science series volume 71, Marcel Dekker,Inc. Another type of soil release polymers is amphiphilic alkoxylatedgrease cleaning polymers comprising a core structure and a plurality ofalkoxylate groups attached to that core structure. The core structuremay comprise a polyalkylenimine structure or a polyalkanolaminestructure as described in detail in WO 2009/087523 (hereby incorporatedby reference). Furthermore random graft co-polymers are suitable soilrelease polymers Suitable graft co-polymers are described in more detailin WO 2007/138054, WO 2006/108856 and WO 2006/113314 (herebyincorporated by reference). Other soil release polymers are substitutedpolysaccharide structures especially substituted cellulosic structuressuch as modified cellulose deriviatives such as those described in EP1867808 or WO 2003/040279 (both are hereby incorporated by reference).Suitable cellulosic polymers include cellulose, cellulose ethers,cellulose esters, cellulose amides and mixtures thereof. Suitablecellulosic polymers include anionically modified cellulose, nonionicallymodified cellulose, cationically modified cellulose, zwitterionicallymodified cellulose, and mixtures thereof. Suitable cellulosic polymersinclude methyl cellulose, carboxy methyl cellulose, ethyl cellulose,hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, estercarboxy methyl cellulose, and mixtures thereof.

Anti-Redeposition Agents:

The detergent compositions of the present disclosure may also includeone or more anti-redeposition agents such as carboxymethylcellulose(CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP),polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylicacid, copolymers of acrylic acid and maleic acid, and ethoxylatedpolyethyleneimines. The cellulose based polymers described under soilrelease polymers above may also function as anti-redeposition agents.

Other suitable adjunct materials include, but are not limited to,anti-shrink agents, anti-wrinkling agents, bactericides, binders,carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foamregulators, hydrotropes, perfumes, pigments, sod suppressors, solvents,and structurants for liquid detergents and/or structure elasticizingagents.

Formulation of Detergent Products

The detergent composition may be in any convenient form, e.g., a bar, ahomogenous tablet, a tablet having two or more layers, a pouch havingone or more compartments, a regular or compact powder, a granule, apaste, a gel, or a regular, compact or concentrated liquid. There are anumber of detergent formulation forms such as layers (same or differentphases), pouches, as well as forms for machine dosing unit.

Pouches can be configured as single or multi-compartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition from the pouchprior to water contact. The pouch is made from water soluble film whichencloses an inner volume. Said inner volume can be divided intocompartments of the pouch. Preferred films are polymeric materialspreferably polymers which are formed into a film or sheet. Preferredpolymers, copolymers or derivatives thereof are selected polyacrylates,and water soluble acrylate copolymers, methyl cellulose, carboxy methylcellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, mostpreferably polyvinyl alcohol copolymers and, hydroxyprpyl methylcellulose (HPMC). Preferably the level of polymer in the film forexample PVA is at least about 60%. Preferred average molecular weightwill typically be from about 20,000 to about 150,000. Films can also beof blend compositions comprising hydrolytically degradable and watersoluble polymer blends such as polyactide and polyvinyl alcohol (knownunder the Trade reference M8630 as sold by Chris Craft In. Prod. OfGary, Ind., US) plus plasticisers like glycerol, ethylene glycerol,Propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry cleaning composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components can be different incomposition than compartments containing solids. Ref: (US2009/0011970A1).

Detergent ingredients can be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

A liquid or gel detergent, which is not unit dosed, may be aqueous,typically containing at least about 20% by weight and up to about 95%water, such as up to about about 70% water, up to about about 65% water,up to about about 55% water, up to about about 45% water, up to aboutabout 35% water. Other types of liquids, including without limitation,alkanols, amines, diols, ethers and polyols may be included in anaqueous liquid or gel. An aqueous liquid or gel detergent may containfrom about 0 to about 30% organic solvent. A liquid or gel detergent maybe non-aqueous.

Laundry Soap Bars

The enzymes of the present disclosure may be added to laundry soap barsand used for hand washing laundry, fabrics and/or textiles. The termlaundry soap bar includes laundry bars, soap bars, combo bars, syndetbars and detergent bars. The types of bar usually differ in the type ofsurfactant they contain, and the term laundry soap bar includes thosecontaining soaps from fatty acids and/or synthetic soaps. The laundrysoap bar has a physical form which is solid and not a liquid, gel or apowder at room temperature. The term solid is defined as a physical formwhich does not significantly change over time, i.e. if a solid object(e.g. laundry soap bar) is placed inside a container, the solid objectdoes not change to fill the container it is placed in. The bar is asolid typically in bar form but can be in other solid shapes such asround or oval.

The laundry soap bar may contain one or more additional enzymes,protease inhibitors such as peptide aldehydes (or hydrosulfite adduct orhemiacetal adduct), boric acid, borate, borax and/or phenylboronic acidderivatives such as 4-formylphenylboronic acid, one or more soaps orsynthetic surfactants, polyols such as glycerine, pH controllingcompounds such as fatty acids, citric acid, acetic acid and/or formicacid, and/or a salt of a monovalent cation and an organic anion whereinthe monovalent cation may be for example Na⁺, K⁺ or NH₄ ⁺ and theorganic anion may be for example formate, acetate, citrate or lactatesuch that the salt of a monovalent cation and an organic anion may be,for example, sodium formate.

The laundry soap bar may also contain complexing agents like EDTA andHEDP, perfumes and/or different type of fillers, surfactants e.g.anionic synthetic surfactants, builders, polymeric soil release agents,detergent chelators, stabilizing agents, fillers, dyes, colorants, dyetransfer inhibitors, alkoxylated polycarbonates, suds suppressers,structurants, binders, leaching agents, bleaching activators, clay soilremoval agents, anti-redeposition agents, polymeric dispersing agents,brighteners, fabric softeners, perfumes and/or other compounds known inthe art.

The laundry soap bar may be processed in conventional laundry soap barmaking equipment such as but not limited to: mixers, plodders, e.g a twostage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnelsand wrappers. The present disclosure is not limited to preparing thelaundry soap bars by any single method. The premix of the presentdisclosure may be added to the soap at different stages of the process.For example, the premix containing a soap, an enzyme, optionally one ormore additional enzymes, a protease inhibitor, and a salt of amonovalent cation and an organic anion may be prepared and and themixture is then plodded. The enzyme and optional additional enzymes maybe added at the same time as the protease inhibitor for example inliquid form. Besides the mixing step and the plodding step, the processmay further comprise the steps of milling, extruding, cutting, stamping,cooling and/or wrapping.

Method of Producing the Composition

The present disclosure also relates to methods of producing thecomposition. The method may be relevant for the (storage) stability ofthe detergent composition: e.g. Soap bar premix method WO2009155557.

Uses

The present disclosure is also directed to methods for using thedetergent compositions thereof. The present disclosure may be used forexample in any detergent application which requires the degradation ofxanthan gum.

Use to Degrade Xanthan Gum

Xanthan gum has been use as an ingredient in many consumer productsincluding foods and cosmetics and has found use in the oil industry.Therefore the degradation of xanthan gum can result in improved cleaningprocesses, such as the easier removal of stains containing gums, such asxanthan gum. Thus the present disclosure is directed to the use ofdetergent compositions comprising xanthan lyases as described herein(e.g. variants) to degrade xanthan gum. An embodiment is the use ofdetergent compositions comprising xanthan lyases as described herein(e.g. variants) together with endoglucanase(s) to degrade xanthan gum.Degradation of xanthan gum can preferably be measured using theviscosity reduction assay (e.g., ViPr assay) or alternatively asdescribed in example 4 herein.

GH9 endoglucanase activity may alternatively be measured by assessmentof reducing ends on xanthan gum pre-treated with xanthan lyase using thecolorimetric assay developed by Lever (1972), Anal. Biochem. 47:273-279, 1972. A preferred embodiment is the use of 0.1% xanthan gumpre-treated with xanthan lyase. Degradation of xanthan gum pre-treatedwith xanthan lyase may be determined by calculating difference betweenblank and sample wherein a difference of more than about 0.5 mAU,preferably more than about 0.6 mAU, more preferably more than about 0.7mAU or even more preferably more than about 0.8 mAU shows degradation ofxanthan gum pre-treated with xanthan lyase.

Xanthan lyase activity may alternatively be measured by assessment ofreducing ends on xanthan gum using the colorimetric assay developed byLever (1972), Anal. Biochem. 47: 273-279, 1972. A preferred embodimentis the use of about 0.1% xanthan gum. Degradation of xanthan gum may bedetermined by calculating difference between blank and sample, wherein adifference of more than about 0.1 mAU, preferably more than about 0.15mAU, more preferably more than about 0.2 mAU or even more preferablymore than about 0.25 mAU, shows degradation of xanthan gum.

Xanthan lyase (e.g. variants as described herein) and endoglucanaseactivity may alternatively be measured by assessment of reducing ends onxanthan gum using the colorimetric assay developed by Lever (1972),Anal. Biochem. 47: 273-279, 1972. A preferred embodiment is the use ofabout 0.1% xanthan gum. Degradation of xanthan gum may be determined bycalculating difference between blank and sample wherein a difference ofmore than about 0.4 mAU, preferably more than about 0.5 mAU, morepreferably more than about 0.6 mAU or even more preferably more thanabout 0.8 mAU shows degradation of xanthan gum.

The present disclosure also relates to methods for degrading xanthan gumcomprising applying a detergent composition comprising one or morexanthan lyases described herein (e.g. variants) to xanthan gum. Thepresent disclosure further relates to methods for degrading xanthan gumcomprising applying a detergent composition comprising one or morexanthan lyases to xanthan gum. An embodiment is a method for degradingxanthan gum comprising applying a detergent composition comprising oneor more xanthan lyases as described herein (e.g. variants) together withone or more endoglucanases to xanthan gum.

Use in Detergents

The present disclosure inter alia relates to the use of detergentcompositions comprising xanthan lyases as described herein (e.g.variants) in cleaning processes such as the laundering of textiles andfabrics (e.g., household laundry washing and industrial laundrywashing), as well as household and industrial hard surface cleaning,such as dish wash. For this, the xanthan lyases (e.g. variants) may beadded to a detergent composition comprising of one or more detergentcomponents.

In some aspects xanthan lyases as described herein (e.g. variants) maybe used together with an endoglucanase(s) in detergent compositions forcleaning processes such as the laundering of textiles and fabrics (e.g.household laundry washing and industrial laundry washing), as well ashousehold and industrial hard surface cleaning, such as dish wash. Forthis, the xanthan lyases (e.g. variants) together with anendoglucanase(s) may be added to a detergent composition comprising ofone or more detergent components.

The polypeptides described herein (e.g. variants) may be added to andthus become a component of a detergent composition. The detergentcomposition may be formulated, for example, as a hand or machine laundrydetergent composition for both household and industrial laundrycleaning, including a laundry additive composition suitable forpre-treatment of stained fabrics and a rinse added fabric softenercomposition, or be formulated as a detergent composition for use ingeneral household or industrial hard surface cleaning operations, or beformulated for hand or machine (both household and industrial)dishwashing operations. In a specific aspect, the present disclosurerelates to a detergent additive comprising a polypeptide as describedherein.

The present disclosure also relates to methods for degrading xanthan gumon the surface of a textile or hard surface, such as dish wash,comprising applying a detergent composition comprising one or morexanthan lyases as described herein (e.g. variants) to xanthan gum. Insome aspects the present disclosure relates to a method for degradingxanthan gum on the surface of a textile or hard surface, such as dishwash, comprising applying a detergent composition comprising one or morexanthan lyases as described herein (e.g. variants) together with one ormore endoglucanases to xanthan gum. In some aspects the presentdisclosure relates to a detergent composition comprising one or moredetergent components as described herein. Also encompassed is the use ofxanthan lyases (e.g. variants) having an enzyme detergency benefit insaid detergent compositions.

It has been contemplated that the use of a xanthan lyase as describedherein (e.g., a variant) alone gives an enzyme detergency benefit,preferably an enzyme detergency benefit on xanthan gum.

In some aspects the present disclosure relates to the use of a detergentcomposition comprising one or more detergent components and an isolatedxanthan lyase as described herein (e.g. a variant) together with a GH9endoglucanase. In some aspects the present disclosure relates to the useof a detergent composition comprising one or more detergent componentsand an isolated xanthan lyase (e.g. a variant described herein) togetherwith a GH9 endoglucanase.

The present disclosure is further defined in the following paragraphs:

1. A detergent composition comprising a xanthan lyase variant,comprising an alteration (e.g., a substitution, deletion or insertion)at one or more positions in a region selected from the group of:

-   -   i) region 1 corresponding to amino acids 154 to 176 of SEQ ID        NO: 2,    -   ii) region 2 corresponding to amino acids 614 to 658 of SEQ ID        NO: 2,    -   iii) region 3 corresponding to amino acids 731 to 803 of SEQ ID        NO: 2,    -   iv) region 4 corresponding to amino acids 807 to 846 of SEQ ID        NO: 2,    -   v) region 5 corresponding to amino acids 872 to 885 of SEQ ID        NO: 2,    -   vi) region 6 corresponding to amino acids 903 to 1004 of SEQ ID        NO: 2,        -   wherein said variant has at least about 60%, e.g., at least            about 65%, at least about 70%, at least about 75%, at least            about 80%, at least about 85%, at least about 90%, at least            about 95%, at least about 96%, at least about 97%, at least            about 98%, or at least about 99%, and less than about 100%            sequence identity to SEQ ID NO: 2; preferably said xanthan            lyase variant has activity on xanthan gum, further            preferably said activity is a xanthan gum degrading            activity.

2. The detergent composition of paragraph 1, wherein the xanthan lyasevariant is a variant of a parent xanthan lyase selected from the groupof:

-   -   a) a polypeptide having at least about 60% sequence identity to        the mature polypeptide of SEQ ID NO: 2;    -   b) a polypeptide encoded by a polynucleotide that hybridizes        under low stringency conditions with (i) the mature polypeptide        coding sequence of SEQ ID NO: 1, or (ii) the full-length        complement of (i);    -   c) a polypeptide encoded by a polynucleotide having at least        about 60% identity to the mature polypeptide coding sequence of        SEQ ID NO: 1; and    -   d) a fragment of the mature polypeptide of SEQ ID NO: 2, which        has xanthan lyase activity.

3. The detergent composition of paragraph 2, wherein the parent xanthanlyase has at least about 60%, e.g., at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99% or about 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 2.

4. The detergent composition of any of paragraphs 2-3, wherein theparent xanthan lyase is encoded by a polynucleotide that hybridizesunder low stringency conditions, medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1 or (ii) the full-length complement of (i).

5. The detergent composition of any of paragraphs 2-4, wherein theparent xanthan lyase is encoded by a polynucleotide having at leastabout 60%, e.g., at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or about 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1.

6. The detergent composition of any of paragraphs 2-5, wherein theparent xanthan lyase comprises or consists of the mature polypeptide ofSEQ ID NO: 2.

7. The detergent composition of any of paragraphs 2-6, wherein theparent xanthan lyase is a fragment of the mature polypeptide of SEQ IDNO: 2, wherein the fragment has xanthan lyase activity.

8. The detergent composition of any of paragraphs 2-7, wherein thexanthan lyase variant has at least about 60%, e.g., at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95% identity, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%, but less than about 100%, sequence identity to the amino acidsequence of the parent xanthan lyase.

9. The detergent composition of any of paragraphs 1-8, wherein saidregion selected from the group of regions 1-6 is a chelator-inducedinstability region;

preferably said chelator-induced instability region (e.g., of SEQ ID NO:2 or another parent xanthan lyase) has one or more of the followingfeatures:

-   -   i) in the presence of a chelator it is less conformationally        stable than one or more or all of its adjacent regions; and/or    -   ii) in the presence of a chelator it is more exposed to said        chelator than one or more or all of its adjacent regions; and/or    -   iii) in the presence of a chelator it is more accessible to said        chelator than one or more or all of its adjacent regions; and/or    -   iv) in the presence of a chelator it is more conformationally        dynamic than one or more or all of its adjacent regions; and/or    -   v) in the presence of a chelator it is more receptive to        deuterium incorporation than one or more or all of its adjacent        regions;    -   further preferably said adjacent region is selected from the        group of:    -   vi) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO:        2,    -   vii) region 8 corresponding to amino acids 177 to 613 of SEQ ID        NO: 2,    -   viii) region 9 corresponding to amino acids 659 to 730 of SEQ ID        NO: 2,    -   ix) region 10 corresponding to amino acids 804 to 806 of SEQ ID        NO: 2,    -   x) region 11 corresponding to amino acids 847 to 871 of SEQ ID        NO: 2,    -   xi) region 12 corresponding to amino acids 886 to 902 of SEQ ID        NO: 2, and    -   xii) region 13 corresponding to amino acids 1005 to 1037 of SEQ        ID NO: 2, further most preferably said chelator is EDTA or        citrate.

10. The detergent composition of any of paragraphs 1-9, wherein in anaqueous solution comprising a detergent component said region (e.g., ofSEQ ID NO: 2 or another parent xanthan lyase) selected from the group ofregions 1-6 is less conformationally stable than one or more or all ofits adjacent regions;

preferably said adjacent region is selected from the group of:

-   -   i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO:        2,    -   ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID        NO: 2,    -   iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID        NO: 2,    -   iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID        NO: 2,    -   v) region 11 corresponding to amino acids 847 to 871 of SEQ ID        NO: 2,    -   vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID        NO: 2, and    -   vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ        ID NO: 2,    -   further preferably said detergent component comprises a        chelator; further most preferably said chelator is EDTA or        citrate.

11. The detergent composition of any of paragraphs 1-10, wherein in anaqueous solution comprising a detergent component said region (e.g., ofSEQ ID NO: 2 or another parent xanthan lyase) selected from the group ofregions 1-6 is more exposed to said detergent component than one or moreor all of its adjacent regions;

preferably said adjacent region is selected from the group of:

-   -   i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO:        2,    -   ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID        NO: 2,    -   iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID        NO: 2,    -   iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID        NO: 2,    -   v) region 11 corresponding to amino acids 847 to 871 of SEQ ID        NO: 2,    -   vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID        NO: 2, and    -   vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ        ID NO: 2,    -   further preferably said detergent component comprises a        chelator; further most preferably said chelator is EDTA or        citrate.

12. The detergent composition of any of paragraphs 1-11, wherein in anaqueous solution comprising a detergent component said region (e.g., ofSEQ ID NO: 2 or another parent xanthan lyase) selected from the group ofregions 1-6 is more accessible to said detergent component than one ormore or all of its adjacent regions;

preferably said adjacent region is selected from the group of:

-   -   i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO:        2,    -   ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID        NO: 2,    -   iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID        NO: 2,    -   iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID        NO: 2,    -   v) region 11 corresponding to amino acids 847 to 871 of SEQ ID        NO: 2,    -   vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID        NO: 2, and    -   vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ        ID NO: 2,    -   further preferably said detergent component comprises a        chelator; further most preferably said chelator is EDTA or        citrate.

13. The detergent composition of any of paragraphs 1-12, wherein in anaqueous solution comprising a detergent component said region (e.g., ofSEQ ID NO: 2 or another parent xanthan lyase) selected from the group ofregions 1-6 is more conformationally dynamic than one or more or all ofits adjacent regions;

preferably said adjacent region is selected from the group of:

-   -   i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO:        2,    -   ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID        NO: 2,    -   iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID        NO: 2,    -   iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID        NO: 2,    -   v) region 11 corresponding to amino acids 847 to 871 of SEQ ID        NO: 2,    -   vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID        NO: 2, and    -   vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ        ID NO: 2,    -   further preferably said detergent component comprises a        chelator; further most preferably said chelator is EDTA or        citrate.

14. The detergent composition of any of paragraphs 1-13, wherein in anaqueous solution comprising a detergent component said region (e.g., ofSEQ ID NO: 2 or another parent xanthan lyase) selected from the group ofregions 1-6 is more receptive to deuterium incorporation than one ormore or all of its adjacent regions;

preferably said adjacent region is selected from the group of:

-   -   i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO:        2,    -   ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID        NO: 2,    -   iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID        NO: 2,    -   iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID        NO: 2,    -   v) region 11 corresponding to amino acids 847 to 871 of SEQ ID        NO: 2,    -   vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID        NO: 2, and    -   vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ        ID NO: 2,    -   further preferably said detergent component comprises a        chelator; further most preferably said chelator is EDTA or        citrate.

15. The detergent composition of any of paragraphs 1-14, the xanthanlyase variant comprising an alteration (e.g., a substitution, deletionor insertion) at one or more positions in two or more regions selectedfrom the group of:

-   -   i) region 1 corresponding to amino acids 154 to 176 of SEQ ID        NO: 2,    -   ii) region 2 corresponding to amino acids 614 to 658 of SEQ ID        NO: 2,    -   iii) region 3 corresponding to amino acids 731 to 803 of SEQ ID        NO: 2,    -   iv) region 4 corresponding to amino acids 807 to 846 of SEQ ID        NO: 2,    -   v) region 5 corresponding to amino acids 872 to 885 of SEQ ID        NO: 2,    -   vi) region 6 corresponding to amino acids 903 to 1004 of SEQ ID        NO: 2,        -   wherein said variant has at least about 60%, e.g., at least            about 65%, at least about 70%, at least about 75%, at least            about 80%, at least about 85%, at least about 90%, at least            about 95%, at least about 96%, at least about 97%, at least            about 98%, or at least about 99%, and less than about 100%            sequence identity to SEQ ID NO: 2, preferably said variant            has activity on xanthan gum, further preferably said            activity is a xanthan gum degrading activity.

16. The detergent composition of any of paragraphs 1-15, wherein saidvariant has at least about 61%, at least about 62%, at least about 63%,at least about 64%, at least about 65%, at least about 66%, at leastabout 67%, at least about 68%, at least about 69%, at least about 70%,at least about 71%, at least about 72%, at least about 73%, at leastabout 74%, at least about 75%, at least about 76%, at least about 77%,at least about 78%, at least about 79%, at least about 80%, at leastabout 81%, at least about 82%, at least about 83%, at least about 84%,at least about 85%, at least about 86%, at least about 87%, at leastabout 88%, at least about 89%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% sequence identity to SEQ ID NO: 2.

17. The detergent composition of any of paragraphs 1-16, wherein saidalteration (e.g., a substitution, deletion or insertion) at one or morepositions is selected from the group of alterations in positions: 155,159, 620, 624, 626, 631, 635, 645, 649, 650, 656, 738, 745, 746, 748,752, 753, 754, 757, 764, 769, 774, 775, 777, 779, 782, 785, 786, 789,792, 796, 799, 800, 801, 819, 824, 843, 845, 875, 903, 911, 912, 915,919, 921, 923, 925, 927, 928, 930, 932, 933, 941, 966, 967, 991 and 998.of SEQ ID NO: 2, wherein numbering is according to SEQ ID NO: 2,preferably alterations in positions: 775, 779 or 923, wherein numberingis according to SEQ ID NO: 2.

18. The detergent composition of any of paragraphs 1-17, wherein saidalteration at one or more positions is selected from the group of:Y155E, A159P, K620R, A624E, A626G, T631N, T631E, S635E, S635T, S635Q,A645S, T649V, T649K, T649R, Q650G, I656V, G738L, K745R, F746L, L748T,P752R, P752K, G753E, G753Q, G753S, S754E, S754L, S754Q, S754R, S757D,S757P, S757E, P764V, P764K, A769D, A769T, A769R, A769S, A769E, A769Q,A769*, A774V, L775M, L775Y, L775A, L775I, L775S, L775F, L775Q, D777K,D777R, P779V, Y782I, A785T, N786K, G789R, K792W, K792Y, K792V, K792A,N796Q, A799H, V800P, D801G, K819R, K819T, K824R, A843P, D845E, K875T,K875E, T903A, T903Q, A911V, A911M, A911S, A912T, A912I, A912Y, T915Q,T915S, T915V, T915A, T919F, T919G, T919D, T921R, T921S, T923H, T923D,T925Q, T925D, T925R, T927K, D928W, Y930H, Y930L, Y930F, A932P, D933M,G941E, G941D, A966P, A967D, N991D and V998K, wherein numbering isaccording to SEQ ID NO: 2.

19. The detergent composition of any of paragraphs 1-18, furthercomprising an alteration at one or more positions in at least one regionselected from the group of:

-   -   vii) region 7 corresponding to amino acids 1 to 153 of SEQ ID        NO: 2,    -   viii) region 8 corresponding to amino acids 177 to 613 of SEQ ID        NO: 2,    -   ix) region 9 corresponding to amino acids 659 to 730 of SEQ ID        NO: 2,    -   x) region 10 corresponding to amino acids 804 to 806 of SEQ ID        NO: 2,    -   xi) region 11 corresponding to amino acids 847 to 871 of SEQ ID        NO: 2,    -   xii) region 12 corresponding to amino acids 886 to 902 of SEQ ID        NO: 2, and    -   xiii) region 13 corresponding to amino acids 1005 to 1037 of SEQ        ID NO: 2.

20. The detergent composition of paragraph 19, wherein said alterationat one or more positions in at least one region selected from the groupof regions 7, 8, 9, 10, 11, 12 and 13 is an alteration at one or morepositions selected from the group of: 9, 15, 18, 46, 58, 66, 89, 95,100, 106, 109, 183, 188, 190, 203, 204, 221, 229, 234, 238, 240, 242,243, 257, 258, 284, 291, 293, 316, 317, 320, 324, 329, 333, 339, 341,352, 354, 360, 372, 377, 399, 400, 419, 440, 450, 451, 454, 458, 481,492, 505, 533, 567, 568, 576, 578, 579, 582, 664, 672, 703, 722, 726,727, 728, 851, 855, 856, 867, 887, 892, 899, 900, 901, 902, 915, 1008and 1016 of SEQ ID NO: 2.

21. The detergent composition of paragraph 20, wherein said alterationat one or more positions in at least one region selected from the groupof regions 7, 8, 9, 10, 11, 12 and 13 comprises one or moresubstitutions selected from the group of: K9R, N15T, T18D, L46D, A58L,S66H, Q89Y, K95E, S100D, N106Y, Q109R, Q109D, Q109F, Q109K, Q109A,K183Q, K183R, V188I, A190Q, A203P, K204R, A221P, E229N, E229S, E229V,I234V, I238W, I238L, I238M, 1240W, N242S, G243V, Y257W, R258E, R284G,K291R, A293G, A293P, K316R, R317K, K320R, L324Q, K329R, K333R, L339M,I341P, V352I, S354P, K360G, K360R, Q372H, F377Y, N399K, K400R, F419Y,N440K, D450P, K451E, K451R, A454V, D458S, K481R, A492H, A492L, T5051,L533I, K567R, G568A, S578K, S578N, S578R, S579R, S579K, S582K, T664K,N672D, I703L, I722F, P726Q, T727P, M728V, S851F, K855R, E856D, P867S,K887R, N892Y, N892W, N892F, G899S, 1900G, D901A, T902F, N1008D andK1016T of SEQ ID NO: 2.

22. The detergent composition of any of paragraphs 1-21, the xanthanlyase variant comprising an alteration at one or more positions selectedfrom the group of positions 624, 631, 635, 649, 656, 738, 752, 753, 754,757, 769, 775, 777, 800, 801, 843, 875, 911 and 915, and an alterationat one or more positions selected from the group of positions 89, 100,190, 229, 234, 352, 360, 399, 440, 458, 492, 567, 582, 664, 672, 703,728, 892, 1008 and 1016 of SEQ ID NO: 2.

23. The detergent composition of paragraph 22, the xanthan lyase variantcomprising one or more substitutions selected from the group of Q89Y,S100D, A190Q, E229S, I234V, V352I, K360G, N399K, N440K, D458S, A492H,A492L, K567R, S582K, T664K, N672D, I703L, M728V, N892Y N1008D andK1016T, and one or more substitutions selected from the group of A624E,T631N, S635E, T649K, I656V, G738L, P752K, P752R, G753E, S754E, S754R,S757D, A769D, L775A, D777R, V800P, D801G, A843P, K875T, A911V and T915A.

24. The detergent composition of any of paragraphs 1-23, wherein thetotal number of alterations compared to the parent xanthan lyase (e.g.,SEQ ID NO: 2) is between from about 1 and about 20, e.g. between fromabout 1 and about 10 or between from about 1 and about 5, such as about1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about9 or about 10 alterations.

25. The detergent composition of any of paragraphs 1-24, wherein saidactivity on xanthan gum is a xanthan gum degrading activity, preferablysaid xanthan lyase variant has EC 4.2.2.12 activity.

26. The detergent composition of any of paragraphs 1-25, wherein saidvariant has an improved stability in a detergent composition compared toa parent xanthan lyase (e.g., with SEQ ID NO: 2); preferably saiddetergent composition comprises a chelator; further preferably saidchelator is EDTA or citrate.

27. The detergent composition of any of paragraphs 1-26, wherein saidvariant has a half-life improvement factor (HIF) of ≥1.0; preferablysaid variant has a half-life improvement factor (HIF) of >1.0, morepreferably at least 1.2, such as at least about 1.5, e.g. at least about2.0, relative to a parent xanthan lyase, e.g. a xanthan lyase with SEQID NO: 2.

28. The detergent composition of paragraph 27, wherein said half-lifeimprovement factor (HIF) is determined after incubation of said xanthanlyase variant in a detergent composition at about 25° C. for a timeperiod from about 30 min to about 20 hours.

29. The detergent composition of any of paragraphs 1-28, wherein saidvariant is selected from the group of i) the xanthan lyase variants setforth in Table 1 herein, ii) the xanthan lyase variants set forth inTable 2 herein, iii) the xanthan lyase variants set forth in Table 3herein, iv) the xanthan lyase variants set forth in Table 4 herein, v)the xanthan lyase variants set forth in Table 5 herein, vi) the xanthanlyase variants set forth in Table 6 herein, and vii) the xanthan lyasevariants set forth in any of Tables 7, 8, 9, 10, 11, 12 or 13 herein.

30. The detergent composition of any one of paragraphs 1-29, whereinsaid composition comprises one or more detergent components; preferablysaid component comprises a chelator; further preferably said chelator isEDTA or citrate.

31. The composition of any of paragraphs 1-30, further comprising one ormore additional enzymes selected from the group of: endoglucanases,proteases, amylases, lichenases, lipases, cutinases, cellulases, xanthanlyases, xyloglucanases, pectinases, pectin lyases, xanthanases,peroxidases, haloperoxygenases, catalases and mannanases, or any mixturethereof.

32. The composition of any of paragraphs 1-31, wherein said compositionis in form of a bar, a homogenous tablet, a tablet having two or morelayers, a pouch having one or more compartments, a regular or compactpowder, a granule, a paste, a gel, or a regular, compact or concentratedliquid.

34. Use of a detergent composition of any of paragraphs 1-32, whereinsaid use is for degrading xanthan gum.

35. The use of paragraph 34, wherein said xanthan lyase variant has anenzyme detergency benefit.

The present disclosure is further described by the following examplesthat should not be construed as limiting the scope of the presentdisclosure.

EXAMPLES Example 1: Hydrogen Exchange—HDX

Continuous amide Hydrogen/Deuterium (H/D) exchange of the parent xanthanlyase of SEQ ID NO: 2 was initiated by addition of 99.9% deuterated 20mM Tris, 1 mM CaCI2), pH 8 in the absence or presence of 5 mM EDTA to afinal deuterium concentration of 85%. H/D exchange was performed intriplicates at 22° C. at a concentration of 1 μM. At five time pointsranging from 15 seconds to 1 hour the samples were quenched by additionof 1:1 (v/v) ice-cold 6M guanidinium hydrochloride, 300 mM phosphate, pH2.05 to a final pH of 2.6. The quenched samples were immediately frozenand stored at −80° C. until LC-MS analysis. Non-deuterated samples wereprepared following the same procedure, but using protiated buffers.Fully deuterated samples (85% D₂O for the parent xanthan lyase of SEQ IDNO: 2) were prepared by over-night incubation in 99.9% deuterated 6Mguanidinium hydrochloride and quenched in 300 mM phosphate, pH 2.3 to afinal pH of 2.6. To study the reversibility of the effects of EDTA onxanthan lyase H/D exchange was done for 10 minutes on three states ofxanthan lyase (XL): (1) XL; (2) XL+5 mM EDTA; (3) XL XL preincubatedwith first 5 mM EDTA for 30 minutes and second with 10 mM CaCl2 for 30minutes. The samples were quenched as described above.

The quenched samples were loaded into a cooled HDX-UPLC system foronline pepsin digestion using an immobilized pepsin column (Pierce,Rockford, USA). The peptides were desalted using a trap column (WatersVanGuard C18, 1.7 μM, 2.1×5 mm) at a flow of 200 μl/min 0.23% formicacid for 3 min and peptides were separated by reverse phasechromatography (Waters Acquity BEH C18, 1.7 μm, 1×100 mm) using atwo-step gradient from 8-18% in 2 min and 18-40% in 10 min 0.23% formicacid in acetonitrile at a flow of 40 μl/min. Positive electrosprayionization mass spectrometry with ion mobility was performed on thepeptides using a Synapt G2 mass spectrometer (Waters, Milford, USA).

The peptic peptides of the mature parent xanthan lyase of SEQ ID NO: 2were identified by tandem mass spectrometry of non-deuterated samplesusing a combination of data independent (MSe) and data dependentacquisition schemes (DDA) and data-analysis in Protein Lynx GlobalServer v. 2.5. Deuterium incorporation of individual peptides weredetermined in DynamX v. 3.0 or in HXexpress (M. Guttman, D. Weis, J.Engen, K. Lee, J. Am. Soc. Mass. Spectrom. 2013, 24, 1906-1912).Statistical analysis (F-tests and student T-tests) were employed todetermine statistically significant changes in H/D exchange (ΔDX)between the analyzed protein states.

Peptides with significant changes in H/D exchange (ΔDX>0.5) wereselected as identifying chelator-induced instability regions within SEQID NO: 2. The following chelator-induced instability regions wereidentified within xanthan lyase of SEQ ID NO: 2: region 1 correspondingto amino acids 154 to 176 of SEQ ID NO: 2, region 2 corresponding toamino acids 614 to 658 of SEQ ID NO: 2, region 3 corresponding to aminoacids 731 to 803 of SEQ ID NO: 2, region 4 corresponding to amino acids807 to 846 of SEQ ID NO: 2, region 5 corresponding to amino acids 872 to885 of SEQ ID NO: 2, and region 6 corresponding to amino acids 903 to1004 of SEQ ID NO: 2.

Example 2: Construction and Expression of Xanthan Lyase Variants

Xanthan lyase parent gene (i.e., SEQ ID NO: 1) was PCR assembled into alinear cassette containing the promoter system on the upstream and catselection maker on the downstream. To enable chromosomal integration ofthe cassette at the Pel locus of B. subtilis host by homologousrecombination, >2 kb DNA sequence identical to the site of integrationwas included on both the sides of the cassette. Genomic DNA preparedfrom the strain containing xanthan lyase parent gene (SEQ ID NO: 1) wasused as template for generating the site-directed mutants. Mutagenicforward and reverse primers were used to generate an approximately 6 kbPCR fragment. This fragment was used as a megaprimer along with anotherforward primer to amplify >8 kb DNA fragment. This fragment containedthe complete cassette (promoter system, xanthan lyase and cat gene alongwith homologous DNA sequence required for recombination at Pel locus)was used for transformation.

The triple promoter system used in the cassette has been described in WO99/43835 and it consists of promoters from Bacillus licheniformisalpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene(amyQ), and the Bacillus thuringiensis cryIIIA promoter including thestabilizing sequence. Protease signal sequence from Bacillus clausii wasincluded to export the protein out of the cells.

Generated variants of the mature parent xanthan lyase of SEQ ID NO: 2are shown in Tables 1 and 2 below. The presence of the alteration wasconfirmed by sequencing.

TABLE 1 Generated variants of the mature parent xanthan lyase of SEQ IDNO: 2 Regions Alteration Region 1 (amino acids 154 to 176 of SEQ ID NO:2): Y155E A159P Region 2 (amino acids 614 to 658 of SEQ ID NO: 2): A624EA626G T631N T631E S635E S635T S635Q A645S T649V T649K T649R Q650G I656VRegion 3 (amino acids 731 to 803 of SEQ ID NO: 2): G738L K745R F746LL748T P752R P752K G753E G753Q G753S S754E S754L S754Q S754R S757D S757PS757E P764V P764K A769D A769T A769R A769S A769E A769Q A769* A774V L775ML775Y L775A L775I L775S L775F L775Q D777K D777R P779V Y782I A785T N786KG789R K792W K792Y K792V K792A N796Q A799H V800P D801G Region 4 (aminoacids 807 to 846 of SEQ ID NO: 2): K819R K819T K824R A843P D845E Region6 (amino acids 903 to 1004 of SEQ ID NO: 2): T903A T903Q A911V A911MA911S A912T A912I A912Y T915Q T915S T915V T915A T919F T919G T919D T921RT921S T923H T923D T925Q T925D T925R T927K D928W Y930H Y930L Y930F A932PD933M G941E G941D A966P A967D N991D V998K

TABLE 2 Generated variants of the mature parent xanthan lyase of SEQ IDNO: 2 Regions Alteration Region 2 (amino acids 614 to 658 of SEQ ID NO:2): T631N Region 3 (amino acids 731 to 803 of SEQ ID NO: 2): A769D A769TL775A L775F L775I L775M L775Q L775S L775Y P779V K792A K792V K792Y N796QA799H D801G Region 5 (amino acids 872 to 885 of SEQ ID NO: 2): K875TRegion 6 (amino acids 903 to 1004 of SEQ ID NO: 2): T903A T903Q A911MA911V A912I A912T A912Y T915S T915V T919D T919G T921R T921S T923D T923HT925D T925Q T925R T927K D928W Y930F Y930H Y930L D933M G941D V998K

Bacillus organism containing a variant was inoculated in LB brothcontaining chloramphenicol (6 μg/ml) and grown overnight at 37° C. Forexpression of xanthan lyase variants, 2% of overnight culture was addedto 300 ml of 10-R medium in 1000 ml baffled flask and grown at 30° C.for 96 hrs. at 180 rpm.

10-R medium contained 33 g/L Soluble starch, 6 g/L (NH4)2HPO4, 5 g/LPotato peptone, 1.2 g/L (MgSO4×7H2O), 12 g/L KH2PO4, 5 g/L(Na2HPO4×2H2O), 18 mL/L of Trace metal solution, 1.8 g/L K2SO4 and 0.1g/L (CaCl2×2H2O) and 0.5 mL/L SB2121 (anti-foam agent). Trace metalsolution was made by mixing 0.49 g/L (MnSO4×H2O), 1.97 g/L (FeSO4×7H2O),0.1 g/L (CuSO4×5H2O), 0.3 g/L ZnCl2 and 19.6 g/L citric acid.

Example 3: Purification of Xanthan Lyase Variants

Prior to purification, Bacillus subtilis broth was clarified bycentrifuging at 8000×g for 30 minutes at 10° C. followed by vacuumfiltration using a combination of Seitz filter (K250) and WHATMAN glassfilter GF/F grade in a Buchner funnel. Finally, the supernatant wasfiltered through 0.22μ Tangential flow filtration unit.

Xanthan lyase variants were purified using three-step automated tandemcolumn chromatography. Macro-Prep Methyl HIC column was pre-equilibratedwith 50 mM Tris, pH 8.0 containing 1 M (NH4)2SO4 and 1 mM CaCl2 buffer.During sample loading onto the column the clarified culture supernatant(250 mL) was diluted 1:1 in-line with 50 mM Tris, pH 8.0 containing 2 M(NH4)2SO4 and) mM CaCl2 buffer to make the final concentration to 1 M.The unbound or weakly bound protein was washed with the equilibrationbuffer until the Absorbance at 280 nm comes below 0.1 AU. Elution wascarried out using 50 mM Tris pH 8 containing 0.5 M (NH4)2SO4 and 1 mMCaCl2. Eluted protein peak was automatically loaded on MEP-Hypercelcolumn pre-equilibrated with 50 mM Tris, pH 8 containing 0.5 M (NH4)2SO4and 1 mM CaCl2. The unbound or weakly bound protein was washed with theequilibration buffer until the Absorbance at 280 nm comes below 0.1 AU.The column was washed again with 50 mM Tris, pH 8 containing 1 mM CaCl2to remove impurities. The Purified protein was eluted with 50 mMNa-acetate, pH 5 containing 1 mM CaCl2. The eluted purified protein wasautomatically transferred to Sephadex G-25 column pre-equilibrated with50 mM MOPS, pH 8 containing 1 mM CaCl₂ for desalting.

Example 4: Detergent Stability Assay

Reagents for the detergent stability assay were prepared as follows:

A stock of 1.0 M MOPS buffer was prepared by dissolving 209.26 g of3-Morpholinopropanesulfonic acid in Milli Q water. pH was adjusted to7.5 using NaOH and the final volume of buffer was made up to 1000 ml.This buffer stock was stored at 4° C. until use. A 50 mM workingsolution of MOPS buffer was prepared by adding 50 ml of 1.0 M stock to950 ml of Milli Q water.

A substrate solution of 0.4% w/v xanthan gum was freshly prepared bydissolving 400 mg of xanthan gum in 100 ml of Milli Q water.

A stock solution mix containing 1.0 M Na2CO3, 0.17 M potassium sodiumtartrate and 5 mM (Bi(NO3)3×5H2O) was prepared by dissolving 106.99 g ofNa2CO3, 47.98 g of potassium sodium tartrate and 2.42 mg of(Bi(NO3)3×5H2O) in Milli Q water for a final volume of 1000 ml. Thisstock solution mix was filtered and stored at room temperature.

A PAHBAH reagent (1.5% PAHBAH) was freshly prepared by dissolving 1.5 gof p-hydroxybenzoic acid hydrazide (PAHBAH) in the stock solution mix.

Detergent Stability Assay:

A. Screening of Culture Supernatants of Variants

The in-detergent stability was determined by measuring the enzymaticactivity present in culture supernatants of variants or wild-typecontrols after incubation with detergent (70%, final concentration) at30° C.

Detergent stress was carried out by addition of 30 μl of culturesupernatant and 70 μl of a Persil Universal Gel detergent (100%) intowells of 96-well microtitre plates which were shaken for 15 min at 1000rpm. Two identical plates were produced whereof one plate was incubatedat 4° C. (unstressed plate) and the other plate was incubated at 30° C.(stressed plate) for 1 hour. After incubation, samples from unstressedand stressed plates were diluted 50× with dilution buffer (50 mM MOPS, 5mM CaCl2, pH 7.5).

To measure the enzyme activity of diluted enzyme-detergent samples,reaction mixtures were prepared in 96-well PCR plates. 50 μl of dilutedsamples were mixed with 50 μl of freshly prepared substrate solution andincubated at 40° C. for 1 hour.

After incubation, 75 μl of PAHBAH reagent was added to reaction mixturein the same PCR plate and incubated in a programmable thermal cycler(T-ROBOT) for 10 min at 90° C. followed by subsequent cooling at 10° C.Samples (25 μl) were transferred to a 384 well microtitre plate and theabsorbance was measured at 405 nm using an Infinite M1000 reader (TECAN,Switzerland).

The residual activity (RA) for variants and wild-type controls wascalculated as the percentage of enzymatic activity remaining afterincubation at 30° C. relative to the enzymatic activity remaining afterincubation at 4° C., i.e., according to the following formula aftersubtracting relevant background absorbance contributions:

Residual activity (RA)=100%*Abs 405 (sample incubated at 30° C.)/Abs405(sample incubated at 4° C.).

The variants with higher detergent stability were picked with respect tothe wild-types grown in the plates.

B. Screening of Purified Variants

The detergent stability of purified variants was determined by measuringthe enzyme activity of the purified protein after incubation withdetergent (90%, final concentration) at 30° C.

Purified variants were diluted to a concentration of 200 ppm using 50 mMMOPS buffer. For detergent treatment, 10 μl of diluted purified sampleswere mixed with 90 μl of Persil Universal Gel detergent (100%) intowells of 96-well microtitre plates which were shaken for 20 min at 1000rpm. Two identical plates were produced whereof one plate was incubatedat 4° C. (unstressed plate) and the other plate was incubated at 30° C.(stressed plate) for 1 hour. After incubation, samples from unstressedand stressed plates were diluted 50× with dilution buffer (50 mM MOPS, 1mM CaCl2, pH 7.5).

Enzymatic activity analysis of unstressed and stressed samples was doneas described in section A.

C. Calculating Half-Lives and Half-Life Improvement Factors (HIF)

Half-life (T½ (in hours)) was calculated at a given detergentconcentration and storage temperature for the wild-type controls and/orvariants, as the degradation follows an exponential decay and theincubation time (hours) is known, i.e., according to the followingformulas:

T½(variant)=(Ln(0.5)/Ln(RA-variant/100))*Time

T½(Wild-type)=(Ln(0.5)/Ln(RA-Wild-type/100))*Time

Where “RA” is the residual activity in percent as calculated above and“Time” is the incubation time in hours.

A half-life improvement factor (HIF) under a given set of storageconditions (detergent concentration and temperature) is calculated asHIF=T½(variant)/T½(Wild-type), where the Wild-type is incubated underthe same storage conditions as the variant.

The obtained HIF values for the purified variants are shown in Table 3below.

TABLE 3 Half-life improvement factors of purified variants: RegionsAlteration HIF — Wild-type 1 Region 1 (amino acids 154 to 176 of SEQY155E 1.4 ID NO: 2): Region 2 (amino acids 614 to 658 of SEQ K620R 1.5ID NO: 2): Q650G 4.8 T631N 1.4 T649V 1.2 Region 3 (amino acids 731 to803 of SEQ K745R 2.4 ID NO: 2): S757D 3.4 G753E 4.4 G753Q 3.8 G753S 2.5S754E 2 P752R 1.4 S754L 1.3 K792W >5 Region 4 (amino acids 807 to 846 ofSEQ K819R 1.2 ID NO: 2): K824R 1.2 Region 6 (amino acids 903 to 1004 ofA966P 1.5 SEQ ID NO: 2): N991D 1.7The obtained HIF values for culture supernatants of variants are shownin Table 4 below.

TABLE 4 Half-life improvement factors of culture supernatants ofvariants: Regions Alteration HIF — Wild-type 1 Region 2 (amino acids 614to 658 of SEQ T631N 1.2 ID NO: 2): Region 3 (amino acids 731 to 803 ofSEQ A769D 3.2 ID NO: 2): A769T 1.5 L775A >5.0 L775F 2.3 L775I >5.0L775M >5.0 L775Q 1.3 L775S >5.0 L775Y >5.0 P779V >5.0 K792A 1.9 K792V3.3 K792Y >5.0 N796Q 1.2 A799H 1.3 D801G 2.5 Region 5 (amino acids 872to 885 of SEQ K875T >5.0 ID NO: 2): Region 6 (amino acids 903 to 1004 ofSEQ T903A 1.3 ID NO: 2): T903Q 1.2 A911M 1.5 A911V 2.1 A912I 1.5 A912T1.6 A912Y 1.4 T915S 1.3 T915V 1.2 T919D 1.2 T919G 1.3 T921R 1.3 T921S1.2 T923D 1.3 T923H 2.3 T925D 1.2 T925Q 1.2 T925R 1.2 T927K 1.2 D928W2.3 Y930F 1.2 Y930H 1.2 Y930L 1.2 D933M 1.5 G941D 1.2 V998K 1.3

Example 5: Half-Life of Xanthan Lyase Variants

Variants of the mature parent xanthan lyase of SEQ ID NO: 2 wereprepared and purified as described above in Examples 2 and 3. Thein-detergent stability of the variants was determined as described inExample 4 by measuring the enzymatic activity present in either culturesupernatants or purified samples of the variants after incubation withdetergent. Incubation was performed using a 70% concentration of PersilUniversal Gel detergent (PUG) at 30° C. for the culture supernatants,and a 70% or 90% concentration of PUG detergent at 30° C. for thepurified variants, with a variant incubation time of one hour for theculture supernatants and one hour or three hours for the purifiedvariants.

The half-lives and calculated half-life improvement factor (HIF) valuesfor culture supernatants are provided in Table 5 below. Table 6 showsthe half-life and half-life improvement factors (HIF) for purifiedvariants, where HIF for variants incubated with a 70% detergentconcentration are calculated based on a wild-type half-life of 0.22 hand variants incubated with a 90% detergent concentration are calculatedbased on a wild-type half-life of 0.20 h.

TABLE 5 Half-life and half-life improvement factor (HIF) of culturesupernatants of variants Half-life Region Mutation (h) HIF — Wild-type0.42 1 Region 1 (amino acids 154 to A159P 0.5 1.2 176 of SEQ ID NO: 2)Region 2 (amino acids 614 to S635T 0.6 1.4 658 of SEQ ID NO: 2) Region 3(amino acids 731 to S754Q 0.5 1.2 803 of SEQ ID NO: 2) S757E 0.6 1.4S757P 0.6 1.4 A769R 0.6 1.4 A769T 1.1 2.6 L775F 1.0 2.4 L775I 1.1 2.6L775Q 0.5 1.2 L775S 1.1 2.6 T778T 0.6 1.4 Y782I 0.7 1.6 N786K 0.5 1.2G789R 0.5 1.2 K792A 0.8 1.9 K792V 1.1 2.6 K792Y 1.1 2.6 N796Q 0.5 1.2A799H 0.5 1.2 Region 5 (amino acids 872 to K875E 0.6 1.4 885 of SEQ IDNO: 2) Region 6 (amino acids 903 to T903A 0.5 1.2 1004 of SEQ ID NO: 2)T903Q 0.5 1.2 A911M 0.6 1.4 A911S 0.6 1.4 A911V 0.9 2.1 A912Y 0.6 1.4A912T 0.7 1.6 T915Q 0.6 1.4 T915S 0.5 1.2 T919D 0.5 1.2 T919F 0.5 1.2T921R 0.6 1.4 T921S 0.5 1.2 T923D 0.5 1.2 T923H 1.0 2.4 T925D 0.5 1.2T925Q 0.5 1.2 T925R 0.5 1.2 T927K 0.5 1.2 D928W 1.0 2.4 Y930F 0.5 1.2Y930H 0.5 1.2 Y930L 0.5 1.2 D933M 0.6 1.4 G941E 0.5 1.2

TABLE 6 Half-life and half-life improvement factor (HIF) of purifiedvariants Detergent Incubation Mutations (%) time (h) Half-life (h) HIFWild-type 70 1 0.22 1 Wild-type 90 1 0.2 1 K620R 70 1 0.6 2.6 T631N 70 10.6 2.5 S635E 90 1 0.7 3.3 S757D 70 1 0.6 2.9 L775A 90 3 1.6 8.1 L775Y90 1 2.2 11 L775M 90 1 3.7 19 P779V 90 1 0.8 4.2 D801G 90 1 0.6 3.1A843P 90 1 0.6 2.9 K875T 90 1 0.7 3.5 T631N, K875T 90 1 1.0 5.1 S757D,D801G 90 1 4.1 20 S757D, K875T 90 3 1.1 5.7 K875T, N991D 90 3 2.1 11

Example 6: Half-Life of Xanthan Lyase Variants with Mutations inChelator-Induced Instability Regions and Adjacent Regions

Variants of the mature parent xanthan lyase of SEQ ID NO: 2 wereprepared and purified as described above in Examples 2 and 3. For thepurposes of this example, variants were produced having mutations in atleast one chelator-induced instability region (regions 1, 2, 3, 4, 5, 6)and in at least one adjacent region (regions 7, 8, 9, 10, 11, 12, 13).The in-detergent stability of the variants was determined as describedin Example 4 by measuring the enzymatic activity present in purifiedsamples of the variants after incubation with detergent. Incubation wasperformed using a 70%, 90% or 95% concentration of Persil Universal Geldetergent (PUG), with incubation at a temperature of 30, 32, 35 or 37°C. and a variant incubation time ranging from one hour to up to 840hours.

Half-lives were calculated as described above in Example 4. In caseswhere the difference in stability between wild-type and variants was toolarge to accurately assess half-life for both wild-type and variantusing the same incubation time, the incubation time for wild-type andvariant is different, e.g. 1 h for wild-type and up to 840 h for themost stable variants.

Further, in order to determine the stability (half-life) within ashorter duration of incubation time for the more stable variants, e.g.<168 h, the incubation temperature for some variants was increased by2-7° C. For variants tested at a higher temperature (i.e. >30° C.), HIFvalues based on the wild-type could not be calculated as the half-lifeof the wild-type (order of magnitude of minutes) could not be determinedaccurately at these temperatures. Stability of the variants in thetables below is therefore reported in terms of half-life (in hours).

Tables 7-13 below show the half-life for the purified variants alongwith information on the test conditions (temperature, detergentconcentration, incubation time) for each variant.

TABLE 7 Half-life of purified variants: Temperature (T) 30° C.,detergent concentration 70% Incubation T Detergent time Half- Mutations(° C.) (%) (h) life (h) No mutations (Wild-type) 30 70 1 0.22 K620R,K855R 30 70 1 1.0 K329R, K745R 30 70 1 0.8 K360R, K745R 30 70 1 0.7A293G, K567R, S579R, K620R 30 70 1 1.5 S100D, N991D 30 70 1 0.5

TABLE 8 Half-life of purified variants: Temperature (T) 30° C.,detergent concentration 90% Incubation T Detergent time Half- Mutations(° C.) (%) (h) life (h) No mutations (Wild-type) 30 90 1 0.20 L339M,K451R, S579R, N672D, K745R, G899S 30 90 1 0.4 V188I, L339M, S579R,N672D, K745R, G899S 30 90 1 0.5 K291R, L339M, S579R, N672D, K745R, G899S30 90 1 0.5 L339M, S579R, T631N, N672D, K745R, G899S 30 90 1 0.5 N242S,L339M, F377Y, S579R, T631N, N672D 30 90 1 0.5 L339M, F377Y, S579R,N672D, K745R, G899S 30 90 1 0.5 F377Y, T631N, K819R, N892Y 30 90 1 0.5A221P, L339M, S579R, N672D, K745R, G899S 30 90 1 0.6 K316R, L339M,S579R, N672D, K745R, G899S 30 90 1 0.6 I238M, L339M, S579R, N672D,K745R, G899S 30 90 1 0.6 S579R, N991D 30 90 1 0.6 K9R, T631N, K819R,N892Y 30 90 1 0.6 S578R, K819R 30 90 1 0.6 S579R, K819R 30 90 1 0.6N242S, L339M, F377Y, S579R, N672D, T727P, N991D 30 90 1 0.6 N242S,L339M, F377Y, S579R, N672D, S757D 30 90 1 0.7 S578R, N991D 30 90 1 0.8A293G, K567R, S579R, K620R 30 90 1 0.8 Q109R, L339M, S579R, N672D,K745R, G899S 30 90 1 0.9 S579R, I722F, T727P, K819R, N892Y 30 90 1 0.9N892Y, N991D 30 90 1 0.9 N15T, N892Y, N991D 30 90 1 1.0 T631N, K819R,N892Y 30 90 1 1.1 K451R, K620R, N892Y 30 90 1 1.1 N672D, K819R, N892Y 3090 3 1.2 S579R, T631N, A645S, N892Y 30 90 1 1.3 K819R, N892Y 30 90 1 1.3S579R, K620R, N892Y 30 90 1 1.3 N672D, K875T 30 90 3 1.3 K316R, S578R,K819R 30 90 3 1.4 N15T, T631N, K819R, N892Y 30 90 3 1.4 K183R, T631N,K819R, N892Y 30 90 3 1.4 S579R, A843P 30 90 3 1.6 S579R, T631N, K819R,N892Y 30 90 3 1.6 E229S, S757D 30 90 3 1.7 R258E, S578R, K819R 30 90 31.7 S579R, S635T 30 90 3 1.8 E229S, K620R 30 90 3 1.8 S579R, K875T 30 903 1.8 S578R, N672D, K819R 30 90 3 1.8 S579R, D801G 30 90 3 1.8 S579R,T727P, N892Y, N991D 30 90 1 1.9 S579R, N892Y, N991D 30 90 3 1.9 A221P,S578R, K819R 30 90 3 1.9 K451R, T631N, N892Y 30 90 1 1.9 N672D, D801G 3090 1 2.0 K291R, S578R, K819R 30 90 3 2.0 I238M, S578R, K819R 30 90 3 2.1S578R, D801G, K819R 30 90 3 2.1 S578R, D801G 30 90 3 2.1 S578R, S757D,K819R 30 90 3 2.2 Q109R, K819R, N892Y 30 90 3 2.2 V188I, S578R, K819R 3090 3 2.2 K9R, S578R, K819R 30 90 3 2.2 L324Q, S578R, K819R 30 90 3 2.3K95E, S578R, K819R 30 90 3 2.3 S578R, K819R, N892Y, N991D 30 90 3 2.3S578R, T631N, K819R, N892Y 30 90 3 2.3 S100D, S578R, N892Y, A967D 30 903 2.3 V188I, S578R, N892Y, A967D 30 90 3 2.3 K291R, S578R, K875T 30 90 32.3 K567R, S578R, K819R 30 90 3 2.4 K316R, S578R, K819R 30 90 3 2.4Q109R, T631N, K819R, N892Y 30 90 3 2.4 Q109R, A843P 30 90 3 2.4 K204R,S578R, K819R 30 90 3 2.4 S578R, K875T 30 90 3 2.4 Q109R, D801G 30 90 32.4 K291R, S578R, N991D 30 90 3 2.4 N106Y, S578R, K819R 30 90 3 2.5Q109R, S635T 30 90 3 2.5 K291R, S578R, D801G 30 90 3 2.5 S578R, K819R,N892Y 30 90 3 2.5 K183R, S578R, K819R 30 90 3 2.5 S578R, K620R, K819R 3090 3 2.6 K291R, S578R, K620R 30 90 3 2.6 S578R, S635T 30 90 3 2.6 S578R,A843P 30 90 3 2.7 K291R, S578R, T631N 30 90 3 2.7 S578R, K819R, K875T 3090 3 2.7 N15T, S578R, K819R 30 90 3 2.8 L339M, S578R, K819R 30 90 3 2.9K291R, S578R, S757D 30 90 3 2.9 Q109R, K875T 30 90 3 2.9 F377Y, S579R,K745R 30 90 3 2.9 Q109R, S578R, K819R 30 90 3 3.7 L339M, S579R, N672D,P726Q, T727P, K745R, A785T, 30 90 1 4.2 N892Y, G899S S100D, R317K,S578R, K620R 30 90 1 4.5 S579R, S757D, N892Y 30 90 1 4.7 K875T, N892Y 3090 3 9.3 N106Y, S578R, K819R, K875T 30 90 16 9.7 N15T, S578R, L775A,K819R 30 90 16 11 Q109R, N672D, K875T 30 90 16 11 Q109R, S757D, K875T 3090 16 11 K183R, S578R, L775A, K819R 30 90 16 11 N106Y, S578R, L775A,K819R 30 90 16 11 N15T, F377Y, S578R, K819R 30 90 16 12 Q109R, D801G,K819R, N892Y 30 90 16 12 K183R, S578R, K819R, K875T 30 90 16 12 Q109R,D801G, K875T 30 90 16 12 Q109R, K819R, K875T, N892Y 30 90 16 12 Q109R,P779V, K819R, N892Y 30 90 16 12 Q109R, P779V, K875T 30 90 16 13 V188I,K291R, S578R, L775A 30 90 20 13 I341P, S578R, L775A 30 90 16 13 Q109R,F377Y, K875T 30 90 16 13 Q109R, S578R, K875T 30 90 16 13 Q109R, S579R,K819R, N892Y 30 90 16 13 K291R, L324Q, S578R, K620R 30 90 20 13 Q109R,T631N, K875T 30 90 16 13 Q109R, S578R, L775A, K819R 30 90 16 13 I341P,S578R, L775M 30 90 16 13 I341P, S578R, T631N 30 90 16 14 Q109R, K875T,N991D 30 90 16 14 Q109R, K620R, K875T 30 90 16 14 K320R, S578R, L775A,K819R 30 90 16 14 Q109R, L775M, K875T 30 90 16 14 Q109R, K875T, N892Y 3090 16 15 E229S, S578R, D801G 30 90 16 15 Q109R, S578R, L775A, K875T 3090 20 15 Q109R, L775M, K819R, N892Y 30 90 16 15 I341P, S578R, K875T 3090 16 16 Q109R, L775A, K819R, N892Y 30 90 16 16 Q109R, S579R, D801G,K875T 30 90 20 16 E229S, S578R, P779V 30 90 16 16 Q109R, L775A, K875T 3090 16 16 Q109R, S578K, K819R, K875T, N892Y 30 90 20 17 E229S, S578R,K875T 30 90 16 17 Q109R, L775A, P779V, K792Y, K819R, N892Y 30 90 20 18E229S, S579R, L775A 30 90 20 18 E229S, S579R, D928W 30 90 20 18 E229S,S578R, L775A 30 90 16 18 E229S, S578R, K819R 30 90 16 18 V188I, S578R,L775A, N892Y, A967D 30 90 16 19 Q109R, E229S, K819R, K875T, N892Y 30 9020 20 Q109R, L775A, P779V, K875T, N892Y 30 90 20 20 Q109R, D801G, K819R,K875T, N892Y 30 90 20 20 E229S, S578R, N991D 30 90 16 20 L339M, S578R,K819R 30 90 20 21 E229S, S579R, K875T 30 90 20 21 Q109R, N892Y, N991D 3090 20 10 K320R, S578R, K819R, K875T 30 90 20 9 K183R, S578R, K819R,N892Y 30 90 20 9 Q109R, S578R, K819R, K875T 30 90 20 14 E229S, S579R,A843P 30 90 20 17 Q109R, S579R, A843P 30 90 20 12 S578R, D801G, A843P 3090 20 10 Q109R, S578K, K875T, N892Y 30 90 20 22 Q109R, L775A, P779V,K792Y, K819R, K875T, N892Y 30 90 20 22 E229S, S578R, K620R 30 90 16 26S578R, K620R, A769# 30 90 3 26 Q109R, L775A, D801G, K875T 30 90 20 26E229S, S578R, T923H 30 90 16 26 Q109R, L775A, K792Y, K819R, N892Y 30 9020 27 Q109R, L775M, K875T, N892Y 30 90 20 27 Q109R, S578K, L775A, K819R,N892Y 30 90 20 27 Q109R, A769T, L775A, K792Y, K819R, N892Y 30 90 20 29Q109R, P779V, K792Y, K875T, N892Y 30 90 20 29 L775A, K875T, N892Y 30 9088 33 Q109R, L775A, P779V, K792Y, K875T, N892Y 30 90 20 33 S578K, L775A,K875T, N892Y, A911V 30 90 90 38 Q109R, L775A, K792Y, K875T 30 90 20 43S578K, P752K, G753E, L775A, K875T, N892Y 30 90 90 46 Q109R, L775A,K792Y, K875T, N892Y 30 90 20 47 S578K, L775A, K875T, N892Y, A912T 30 9090 49 S578K, P752R, G753E, L775A, K875T, N892Y 30 90 90 49 S578K, P752R,G753E, S754E, L775A, K875T, N892Y 30 90 90 51 Q109R, L775A, K875T, N892Y30 90 88 54 S578K, G753E, S754E, L775A, K875T, N892Y 30 90 167 61 S578K,P752K, G753E, S754E, L775A, K875T, N892Y 30 90 167 62 Q109R, E229V,L775A, D801G, K875T 30 90 88 67 S578K, P752R, S754E, L775A, K875T, N892Y30 90 167 67 S578K, A769D, L775A, K875T, N892Y 30 90 90 68 S578K, L775A,D801G, K875T, N892Y 30 90 90 69 Q109R, A769T, L775A, K875T, N892Y 30 90168 71 Q109R, S754E, A769T, L775A, K875T, N892Y 30 90 88 72 S578K,G753E, L775A, K875T, N892Y 30 90 167 73 Q109R, L775A, K875T, N892Y 30 90168 75 Q109R, A769T, L775A, K792Y, K875T, N892Y 30 90 168 78 E229S,G753E, S754E, L775A, K875T, N892Y 30 90 167 79 E229S, P752R, G753E,L775A, K875T, N892Y 30 90 167 80 E229S, P752K, G753E, L775A, K875T,N892Y 30 90 167 81 S100D, L775A, D801G, K875T, N892Y 30 90 167 81 Q109R,A769T, L775A, K875T, N892Y 30 90 168 82 E229S, P752R, G753E, S754E,L775A, K875T, N892Y 30 90 167 82 S754E, L775A, D801G, K875T, N892Y 30 90167 86 E229S, S578R, G753E, N892Y 30 90 88 86 L775A, D801G, K875T, N892Y30 90 168 86 Q109R, S578K, L775A, D801G, K875T 30 90 88 86 E229S, S578R,L775A, N892Y 30 90 88 87 E229S, G753E, L775A, K875T, N892Y 30 90 167 89Q109R, E229S, A769T, L775A, K875T, N892Y 30 90 88 95 Q109R, L775A,P779V, K792Y, D801G, K819R, K875T, 30 90 88 96 N892Y Q109R, G753E,S754E, A769T, L775A, K875T, N892Y 30 90 168 102 Q109R, P752R, G753E,S754E, A769T, L775A, K875T, 30 90 168 102 N892Y E229S, P752K, G753E,S754E, L775A, K875T, N892Y 30 90 167 103 E229S, S578R, G753E, A769D,K792Y, N892Y 30 90 168 106 P752R, S754E, L775A, D801G, K875T, N892Y 3090 167 109 S100D, E229S, S578R, N892Y, A912T 30 90 168 109 E229S, S578R,L775A, P779V, K792Y, N892Y 30 90 168 110 E229S, S578R, P752K, S754E,K792Y, N892Y, A912T 30 90 168 112 E229S, S578R, P752R, G753E, K792Y,N892Y, A912T 30 90 168 116 E229S, L775A, D801G, K875T, N892Y 30 90 167116 Q109R, P752K, G753E, A769T, L775A, K875T, N892Y 30 90 88 117 E229S,S578K, A769D, K792Y 30 90 88 119 Q109R, P752K, G753E, L775A, D801G,K875T 30 90 88 120 Q109R, G753E, A769T, L775A, K875T, N892Y 30 90 88 120E229S, S578R, K792Y, D801G, N892Y 30 90 168 120 E229S, S578K, L775A,P779V, K792Y 30 90 88 122 E229S, S578R, A769D, P779V, K792Y, N892Y 30 90168 122 E229S, A492L, S578R, N892Y, A912T 30 90 168 122 E229S, S578R,A769S, K792Y, N892Y 30 90 168 123 E229S, A769D, L775A, K875T, N892Y 3090 167 125 Q109R, E229S, A769T, L775A, K792Y, K875T, N892Y 30 90 168 125G753E, L775A, D801G, K875T, N892Y 30 90 168 127 E229S, F419Y, S578K,G753E 30 90 168 127 E229S, S578R, A769D, L775A, K792Y, N892Y, A912T 3090 168 127 Q109R, E229S, P752K, G753E, A769T, L775A, K875T, 30 90 168128 N892Y Q109R, E229S, P752K, S754E, A769T, L775A, K875T, 30 90 168 130N892Y Q109R, E229S, G753E, A769T, L775A, K875T, N892Y 30 90 168 130Q109R, E229S, P752R, G753E, S754E, A769T, L775A, 30 90 168 131 K875T,N892Y E229S, S578K, D801G 30 90 88 133 Q109R, P752K, G753E, S754E,A769T, L775A, K875T, 30 90 88 133 N892Y E229S, S578K, G753E, A843P 30 90168 134 E229S, S578R, L775A, K792Y, N892Y, A912T 30 90 168 137 E229S,S578K, T631N, G753E 30 90 168 137 E229S, S578K, P752R, G753E 30 90 88139 Q109R, P752R, G753E, A769T, L775A, K875T, N892Y 30 90 88 139 E229S,A492L, S578K, G753E, D801G 30 90 168 140 Q109R, E229S, G753E, S754E,A769T, L775A, K875T, 30 90 168 141 N892Y E229S, S578K, K875T 30 90 88143 E229S, S578R, G753E, A769D, L775A, N892Y 30 90 167 143 G753E, L775A,D801G, K875T, N892Y 30 90 167 144 E229S, S578K, G753E, N892Y 30 90 168144 E229N, S578K, A769D, L775A, K875T, N892Y 30 90 168 145 E229S, S578K,G753E, L775A, P779V 30 90 168 145 Q109R, E229S, S578K, A769T, L775A,K875T, N892Y 30 90 168 146 E229S, S578R, P752K, K792Y, N892Y, A912T 3090 168 149 E229S, K360R, S578K, P752K, G753E, S754E 30 90 168 149 E229S,A492L, S578K, G753E 30 90 168 150 E229S, S578K, K792Y, N892Y 30 90 168151 E229S, S578K, P779V 30 90 88 151 E229S, S578K, G753E 30 90 88 153E229S, K360R, S578K, A769D, L775A, K792Y 30 90 168 154 Q109R, E229S,T631N, G753E, S754E, A769T, L775A, 30 90 168 156 K875T, N892Y Q109R,E229S, A769D, L775A, K875T, N892Y 30 90 168 156 G753E, L775A, D801G,K875T, N892Y, A912T 30 90 168 157 Q109R, E229S, K567R, G753E, S754E,A769T, L775A, 30 90 168 158 K875T, N892Y P752R, G753E, L775A, D801G,K875T, N892Y 30 90 167 158 E229S, S578K, T631N, P752K, G753E, S754E 3090 168 159 E229S, A492L, S578K, G753E, K1016T 30 90 168 159 E229S,S578K, T631N, G753E, S754E 30 90 168 159 E229S, S578K, P752R 30 90 88160 E229S, S578R, G753E, A769D, P779V, N892Y 30 90 168 160 E229S, S578K,G753E, N1008D 30 90 168 162 P752K, G753E, L775A, D801G, K875T, N892Y 3090 167 162 E229S, S578K, G753E, A912T 30 90 168 163 E229S, S578K, G753E,S754E 30 90 88 165 E229S, S578K, P752R, S754E 30 90 88 165 E229S, A492L,S578K, G753E, S754E 30 90 168 167 E229S, S578K, A769D, P779V 30 90 88168 A769D, L775A, D801G, K875T, N892Y 30 90 167 172 Q109R, E229S, G753E,S754E, A769T, L775A, A843P, 30 90 168 172 K875T, N892Y Q109R, E229S,S635T, G753E, S754E, A769T, L775A, 30 90 168 175 K875T, N892Y E229S,S578K, T631N, G753E, D801G 30 90 168 176 E229S, K360R, S578K, P752R,S754E, A769T, L775A, 30 90 168 178 K875T, N892Y E229S, S578K, T923H 3090 88 178 Q109R, E229S, G753E, S754E, A769T, L775A, K875T, 30 90 168 180N892Y, K1016T G753E, L775A, D801G, K875T, N892Y, V998K 30 90 168 180E229S, S578K, T631N, G753E, K1016T 30 90 168 180 Q109R, E229S, S578K,G753E, S754E, A769T, L775A, 30 90 168 182 K875T, N892Y E229S, S578K,L775A, K792Y 30 90 88 184 E229S, S578K, P752K, S754E 30 90 88 184 E229S,S578K, A911V, A912T, T923H 30 90 88 186 E229S, S578K, T631N, G753E,A769T, L775A 30 90 168 187 E229S, S578K, T631N, G753E, A769D, K792Y 3090 168 188 E229S, S578K, P752K 30 90 88 188 P752K, G753E, S754E, L775A,D801G, K875T, N892Y 30 90 168 190 Q109R, E229S, G753E, S754E, A769T,L775A, K875T, 30 90 168 191 N892Y, A932P E229S, A492L, S578K, G753E,L775A 30 90 168 195 P752K, G753E, S754E, L775A, D801G, K875T, N892Y 3090 167 195 E229S, S578K, T631N, G753E, D901A 30 90 168 197 E229S, K360R,S578K, P752R, A769D, L775A, K875T, 30 90 167 209 N892Y E229S, S578K,A911V 30 90 88 210 Q109R, E229S, A769T, L775A, D801G, K875T, N892Y 30 90168 221 E229S, A492L, S578K, G753E, N1008D 30 90 168 226 E229S, A492L,S578K, G753E, L775A, P779V 30 90 168 227 E229S, A492L, S578K, G753E,A769D 30 90 168 230 E229S, S578K, T631N, G753E, A769D, A774V, L775A, 3090 168 234 P779V, K792Y A769D, L775A, D801G, K875T, N892Y, V998K 30 90168 238 E229S, K360R, S578K, S754E, A769D, L775A, K875T, 30 90 168 238N892Y E229S, S578K, A769D, L775A, K875T, N892Y 30 90 168 239 S635T,A769D, L775A, D801G, K875T, N892Y 30 90 168 243 E229S, S578K, S754E 3090 88 243 Q109R, E229S, G753E, S754E, A769E, L775A, K875T, 30 90 168 249N892Y E229S, A492L, S578K, G753E, L775A, K792Y 30 90 168 259 A769D,L775A, D801G, A843P, K875T, N892Y 30 90 168 270 G753E, S754E, L775A,D801G, K875T, N892Y 30 90 168 276 G753E, S754E, A769D, L775A, D801G,K875T, N892Y 30 90 168 277 E229S, K360R, S578K, P752K, A769D, L775A,K875T, 30 90 168 280 N892Y E229S, S578K, T631N, G753E, A912T 30 90 168281 Q109R, E229S, N672D, G753E, S754E, A769T, L775A, 30 90 168 297K875T, N892Y A190Q, A769D, L775A, D801G, K875T, N892Y 30 90 168 297P752R, G753E, A769D, L775A, D801G, K875T, N892Y 30 90 168 300 P752R,G753E, S754E, A769D, L775A, D801G, K875T, 30 90 168 341 N892Y A769D,L775A, D801G, K875T, N892Y, N1008D 30 90 168 343 G753E, A769D, L775A,D801G, K875T, N892Y 30 90 168 344 E229S, K360R, S578K, P752R, G753E,A769D, L775A, 30 90 168 375 K875T, N892Y N672D, A769D, L775A, D801G,K875T, N892Y 30 90 168 380 N672D, G753E, L775A, D801G, K875T, N892Y 3090 168 >385 Q109R, G753E, S754E, A769T, L775A, D801G, K875T, 30 90168 >385 N892Y E229S, G753E, L775A, D801G, K875T, N892Y 30 90 168 >385E229N, G753E, L775A, D801G, K875T, N892Y 30 90 168 >385 P752K, G753E,S754E, A769D, L775A, D801G, K875T, 30 90 168 >385 N892Y E229N, A769D,L775A, D801G, K875T, N892Y 30 90 168 >385 E229S, K360R, S578K, P752K,G753E, A769D, L775A, 30 90 168 >385 K875T, N892Y E229S, A769D, L775A,D801G, K875T, N892Y 30 90 168 >385 E229S, K360R, S578K, G753E, S754E,A769D, L775A, 30 90 168 >385 K875T, N892Y Q109R, N672D, G753E, S754E,A769T, L775A, K875T, 30 90 168 >385 N892Y T631N, A769D, L775A, D801G,K875T, N892Y 30 90 168 >385 Q109R, E229S, G753E, S754E, A769T, L775A,D801G, 30 90 168 >385 K875T, N892Y

TABLE 9 Half-life of purified variants: Temperature (T) 30° C.,detergent concentration 95% Incubation Half- T Detergent time lifeMutations (° C.) (%) (h) (h) No mutations (Wild-type) 30 95 1 <0.2E229N, N672D, P752K, G753E, A769D, L775A, D801G, 30 95 672 281 K875T,N892Y Q109R, E229S, N672D, P752R, G753E, S754E, A769T, 30 95 672 310L775A, D801G, K875T, N892Y Q109R, E229S, N672D, P752R, G753E, S754E,A769T, 30 95 672 316 L775A, D801G, K875T, N892Y, D901A Q109R, A159P,E229S, N672D, P752R, G753E, S754E, 30 95 672 355 A769T, L775A, D801G,K875T, N892Y E229N, N672D, P752R, G753E, A769D, L775A, D801G, 30 95 672355 K875T, N892Y E229N, N672D, G753E, S754E, A769D, L775A, D801G, 30 95672 356 K875T, N892Y, N1008D Q109R, E229S, G753E, S754E, A769D, L775A,D801G, 30 95 672 357 K875T, N892Y, N1008D Q109R, E229S, G753E, S754E,A769D, L775A, D801G, 30 95 672 358 K875T, N892Y Q109R, E229S, N672D,G753E, S754E, A769T, L775A, 30 95 672 359 D801G, K875T, N892Y, T923HQ109R, A159P, E229S, G753E, S754E, A769D, L775A, 30 95 672 366 D801G,A843P, K875T, N892Y Q109R, A159P, E229S, S635E, T649K, G753E, S754E, 3095 672 373 A769D, L775A, D801G, K875T, N892Y Q109R, A190Q, E229S, N672D,G753E, S754E, A769T, 30 95 672 383 L775A, D801G, K875T, N892Y Q109R,E229S, T631N, N672D, P752R, G753E, S754E, 30 95 672 383 A769T, L775A,D801G, K875T, N892Y E229N, N672D, G753E, S754E, A769D, L775A, D801G, 3095 672 384 K875T, N892Y Q109R, E229S, N672D, I703L, G753E, S754E, A769T,30 95 672 399 L775A, D801G, K875T, N892Y E229N, P752K, G753E, A769D,L775A, D801G, A843P, 30 95 672 404 K875T, N892Y Q109R, E229S, N672D,G753E, S754E, A769E, L775A, 30 95 672 405 D801G, K875T, N892Y Q109R,A159P, E229S, N672D, G753E, S754E, A769T, 30 95 672 421 L775A, D801G,A843P, K875T, N892Y Q109R, A159P, E229S, G753E, S754E, A769D, L775A, 3095 672 428 D801G, K875T, N892Y Q109R, A159P, E229S, I703L, G753E, S754E,A769D, L775A, 30 95 672 433 D801G, K875T, N892Y Q109R, A159P, E229S,K567R, N672D, G753E, S754E, 30 95 672 452 A769T, L775A, D801G, K875T,N892Y Q109R, A159P, E229S, N672D, G753E, S754E, A769T, 30 95 672 454L775A, D801G, K875T, N892Y Q109R, E229S, T631N, N672D, P752R, G753E,S754E, 30 95 672 477 A769D, L775A, D801G, A843P, K875T, N892Y Q109R,A159P, E229S, T631N, G753E, S754E, A769D, 30 95 672 481 L775A, D801G,K875T, N892Y Q109R, E229S, N672D, G753E, S754E, A769T, L775A, 30 95 672485 D801G, A843P, K875T, N892Y Q109R, E229S, N672D, I703L, P752R, G753E,S754E, A769T, 30 95 672 499 L775A, D801G, K875T, N892Y Q109R, E229S,T631N, S635E, T649K, N672D, P752R, 30 95 672 513 G753E, S754E, A769D,L775A, D801G, K875T, N892Y Q109R, A159P, E229S, A624E, A626G, S635E,T649K, 30 95 672 519 N672D, G753E, S754E, S757D, A769T, L775A, D801G,K875T, N892Y Q109R, A159P, E229S, S635E, T649K, N672D, I703L, G753E, 3095 672 529 S754E, S757D, A769T, L775A, D801G, K875T, N892Y Q109R, E229S,T631N, N672D, P752R, G753E, S754E, 30 95 672 562 S757D, A769T, L775A,D801G, K875T, N892Y Q109R, E229S, T631N, N672D, P752R, G753E, S754E, 3095 672 565 S757D, A769D, L775A, D801G, K875T, N892Y E229S, I234V, S635E,T649K, I656V, N672D, G753E, A769D, 30 95 672 611 L775A, D801G, K875T,N892Y Q109R, E229S, T631N, N672D, P752R, G753E, S754E, 30 95 672 637S757D, A769D, L775A, D801G, K875T, N892Y, K1016T E229S, S635E, T649K,I656V, N672D, S754E, A769D, 30 95 672 662 L775A, D801G, K875T, N892YE229S, A624E, S635E, T649K, I656V, N672D, I703L, G753E, 30 95 672 663S754E, A769D, L775A, D801G, K875T, N892Y, N1008D E229S,, N440K,, S582K,N672D, G753E, S754E, A769D, 30 95 672 691 L775A, D801G, K875T, N892Y,N1008D T18D, Q109R, E229S, T631N, N672D, P752R, G753E, S754E, 30 95 840694 S757D, A769D, L775A, D801G, K875T, N892Y E229S, N440K, S582K, T631N,N672D, G753E, S754E, 30 95 840 713 A769D, L775A, D801G, K875T, N892Y,N1008D L46D, E229S, K360G, D458S, S582K, N672D, G753E, S754E, 30 95 840714 A769D, L775A, D801G, K875T, N892Y, N1008D Q109R, E229S, K567R,T631N, N672D, P752R, G753E, 30 95 840 732 S754E, S757D, A769D, L775A,D801G, K875T, N892Y S100D, E229S, S635E, T649K, I656V, N672D, I703L,G753E, 30 95 672 754 A769D, L775A, D801G, A843P, K875T, N892Y, N1008DE229S, D458S, K567R, T631N, N672D, G753E, S754E, 30 95 672 780 A769D,L775A, D801G, K875T, N892Y E229S, D458S, S582K, N672D, G753E, S754E,A769D, 30 95 672 783 L775A, D801G, K875T, N892Y, N1008D E229S, S635E,T649K, I656V, N672D, G753E, S754E, 30 95 672 796 A769D, L775A, D801G,K875T, N892Y E229S, N672D, G753E, S754E, A769D, L775A, D801G, 30 95 672827 K875T, N892Y Q109R, E229S, K360G, T631N, N672D, P752R, G753E, 30 95840 831 S754E, S757D, A769D, L775A, D801G, K875T, N892Y E229N, T631N,N672D, G753E, S754E, A769D, L775A, 30 95 672 834 D801G, K875T, N892Y,N1008D E229S, S635E, T649K, I656V, N672D, P752R, G753E, S754E, 30 95 672836 A769D, L775A, D801G, K875T, N892Y E229S, I234V, N672D, G753E, S754E,A769D, L775A, 30 95 840 844 D801G, K875T, N892Y E229S, S635E, T649K,I656V, N672D, G753E, A769D, 30 95 672 857 L775A, D801G, K875T, N892YE229S, I234V, A492L, S582K, N672D, M728V, G753E, 30 95 840 857 S754E,A769D, L775A, D801G, K875T, N892Y E229S, S635E, T649K, I656V, N672D,G753E, S754E, 30 95 840 868 A769D, L775A, D801G, K875T, N892Y E229S,S635E, T649K, I656V, N672D, G753E, A769D, 30 95 672 943 L775A, D801G,K875T, N892Y A159P, E229S, N440K, N672D, G753E, S754E, A769D, 30 95 672946 L775A, D801G, K875T, N892Y E229S, D458S, N672D, G753E, S754E, A769D,L775A, 30 95 840 950 D801G, K875T, N892Y E229S, D458S, T631N, N672D,G753E, S754E, S757D, 30 95 672 956 A769D, L775A, D801G, K875T, N892YS100D, E229S, S635E, T649K, I656V, N672D, P752K, G753E, 30 95 672 956S754E, A769D, L775A, D801G, K875T, N892Y E229S, D458S, T631E, N672D,G753E, S754E, A769D, 30 95 840 961 L775A, D801G, K875T, N892Y E229S,S582K, N672D, G753E, S754E, A769D, L775A, 30 95 672 966 D801G, K875T,N892Y E229S, S635E, T649K, I656V, N672D, P752K, G753E, S754E, 30 95 672978 A769D, L775A, D801G, K875T, N892Y E229S, D458S, S582K, T631E, N672D,G753E, S754E, 30 95 840 993 A769D, L775A, D801G, K875T, N892Y A190Q,E229S, S635E, T649K, I656V, N672D, I703L, G753E, 30 95 672 1004 S754E,S757D, A769D, L775A, D801G, K875T, N892Y, N1008D A190Q, E229S, S635E,T649K, I656V, N672D, I703L, G753E, 30 95 672 1015 S754E, A769D, L775A,V800P, D801G, K875T, N892Y, N1008D A190Q, E229S, I234V, S582K, N672D,G753E, S754E, 30 95 672 1019 A769D, L775A, D801G, K875T, N892Y T18D,E229S, S582K, N672D, G753E, S754E, A769D, L775A, 30 95 840 1023 D801G,K875T, N892Y E229S, D458S, T631E, N672D, G753E, S754E, S757D, 30 95 8401030 A769D, L775A, D801G, K875T, N892Y Q89Y, E229S, N672D, G753E, S754E,A769D, L775A, 30 95 840 1038 D801G, K875T, N892Y S100D, E229S, D458S,K567R, S635E, N672D, G753E, 30 95 672 1041 S754E, A769D, L775A, D801G,K875T, N892Y E229S, S635E, T649K, I656V, N672D, I703L, G753E, S754E, 3095 840 1045 A769D, L775A, D801G, K875T, N892Y, N1008D E229S, V352I,S635E, T649K, I656V, N672D, G753E, S754E, 30 95 840 1065 A769D, L775A,V800P, D801G, K875T, N892Y E229S, N672D, P752R, G753E, S754E, A769D,L775A, 30 95 672 1066 D801G, K875T, N892Y E229S, K360G, D458S, S582K,N672D, G753E, S754E, 30 95 840 1067 A769D, L775A, D801G, K875T, N892Y,N1008D E229S, I234V, A492L, N672D, G753E, S754E, A769D, 30 95 840 1070L775A, D777R, D801G, K875T, N892Y E229S, S635E, T649K, N672D, G753E,S754E, A769D, 30 95 840 1081 L775A, D801G, K875T, N892Y, N1008D E229S,N440K, S582K, A624E, N672D, P752R, G753E, 30 95 624 1087 S754E, A769D,L775A, D801G, K875T, N892Y, N1008D E229S, I234V, S582K, N672D, G753E,S754E, A769D, 30 95 792 1094 L775A, V800P, D801G, K875T, N892Y T18D,E229S, S582K, N672D, G753E, S754E, A769D, L775A, 30 95 672 1101 D801G,K875T, N892Y, T902F Q89Y, E229S, N440K, S582K, A624E, N672D, G753E, 3095 672 1117 S754E, A769D, L775A, D801G, K875T, N892Y E229S, D458S,T631E, N672D, G753E, S754E, A769D, 30 95 840 1121 L775A, D777K, D801G,K875T, N892Y E229S, S635E, T649K, I656V, N672D, P752K, G753E, 30 95 6721141 A769D, L775A, D801G, A843P, K875T, N892Y E229S, S635E, T649K,I656V, N672D, G753E, S754E, S757D, 30 95 672 1145 A769D, L775A, D801G,K875T, N892Y E229S, N440K, S582K, N672D, G753E, S754E, A769D, 30 95 6721146 L775A, D801G, A843P, K875T, N892Y, N1008D S100D, E229S, S635E,T649K, I656V, N672D, G753E, S754E, 30 95 672 1153 A769D, L775A, D801G,K875T, N892Y L46D, E229S, K360R, S578K, N672D, G753E, S754E, A769D, 3095 840 1172 L775A, D801G, K875T, N892Y E229S, T631N, N672D, I703L,P752K, G753E, A769D, 30 95 840 1183 L775A, D801G, A843P, K875T, N892YE229S, A624E, S635E, T649K, I656V, N672D, G738L, 30 95 792 1192 G753E,S754R, S757D, A769D, L775A, D777K, D801G, K875T, N892Y E229S, N440K,S582K, A624E, N672D, G753E, S754E, 30 95 624 1214 A769D, L775A, V800P,D801G, K875T, N892Y E229S, D458S, K567R, S635E, N672D, G753E, S754E, 3095 624 1226 A769D, L775A, D801G, K875T, N892Y A190Q, E229S, S635E,T649K, I656V, N672D, I703L, P752R, 30 95 672 1238 G753E, S754E, A769D,L775A, D801G, K875T, N892Y, N1008D E229S, T631N, N672D, I703L, P752K,G753E, S754E, 30 95 840 1259 A769D, L775A, D801G, K875T, N892Y A190Q,E229S, T631N, N672D, I703L, P752K, G753E, 30 95 840 1269 A769D, L775A,D801G, K875T, N892Y A190Q, E229S, S635E, T649K, I656V, N672D, P752K, 3095 672 1282 G753E, A769D, L775A, D801G, A843P, K875T, N892Y A159P,A190Q, E229S, I234V, S582K, N672D, G753E, 30 95 672 1289 S754E, A769D,L775A, D801G, K875T, N892Y E229S, A492L, S635E, T649K, I656V, N672D,P752R, G753E, 30 95 672 1298 S754E, A769D, L775A, D801G, K875T, N892Y,N1008D A190Q, E229S, S582K, N672D, G753E, S754E, A769D, 30 95 792 1299L775A, D801G, K875T, N892Y T18D, E229S, S582K, N672D, G753E, S754E,P764K, A769D, 30 95 624 1299 L775A, D801G, K875T, N892Y E229S, N440K,S582K, N672D, P752R, G753E, S754E, 30 95 624 1328 S757D, A769D, L775A,D801G, K875T, N892Y, N1008D E229S, A492L, S635E, T649K, I656V, N672D,G753E, 30 95 840 1329 A769D, L775A, D801G, K875T, N892Y E229S, S582K,S635E, N672D, P752R, G753E, S754E, 30 95 792 1352 A769D, L775A, D801G,K875T, N892Y, N1008D A190Q, E229S, N440K, S582K, A624E, S635E, N672D, 3095 672 1358 G753E, S754E, A769D, L775A, D801G, K875T, N892Y A190Q,E229S, D458S, T631N, N672D, G753E, S754E, 30 95 624 1398 A769D, L775A,D801G, K875T, N892Y E229S, I234V, A492L, S582K, N672D, G753E, S754E, 3095 840 1426 A769D, L775A, D801G, K875T, N892Y E229S, N440K, S582K,S635E, N672D, G753E, S754E, 30 95 840 1456 A769D, L775A, D801G, A843P,K875T, N892Y, N1008D S100D, A190Q, E229S, I234V, S582K, N672D, G753E, 3095 672 1460 S754E, A769D, L775A, D801G, K875T, N892Y A190Q, E229S,K360G, D458S, S582K, T664K, N672D, 30 95 792 1481 G753E, S754E, A769D,L775A, D801G, K875T, N892Y, N1008D S100D, E229S, N440K, S582K, N672D,P752R, G753E, 30 95 840 1489 S754E, A769D, L775A, D801G, K875T, N892Y,N1008D E229S, S635E, T649K, I656V, N672D, P752K, G753E, S757D, 30 95 6721519 A769D, L775A, D801G, A843P, K875T, N892Y S100D, E229S, K360G,D458S, S582K, N672D, G753E, 30 95 792 1530 S754E, S757D, A769D, L775A,D801G, A843P, K875T, N892Y, T915A, N1008D E229S, N440K, S582K, A624E,S635E, N672D, G738L, 30 95 792 1535 G753E, S754E, S757D, A769D, L775A,D801G, K875T, N892Y S100D, E229S, K360G, D458S, S582K, N672D, G753E, 3095 840 1538 S754E, A769D, L775A, D801G, K875T, N892Y, N1008D A190Q,E229S, D458S, T631N, N672D, G753E, S754E, 30 95 672 1551 A769D, L775A,D801G, A843P, K875T, N892Y A190Q, E229S, K360G, D458S, S582K, N672D,G753E, 30 95 840 1556 S754E, A769D, L775A, D801G, K875T, N892Y, N1008DE229S, S582K, S635E, T649K, I656V, N672D, M728V, 30 95 672 1594 G753E,S754R, S757D, A769D, L775A, D801G, K875T, N892Y A190Q, E229S, N440K,S582K, A624E, S635E, N672D, 30 95 792 1601 G753E, S754E, S757D, A769D,L775A, D801G, K875T, N892Y S100D, A190Q, E229S, S635E, T649K, I656V,N672D, I703L, 30 95 624 1606 G753E, S754E, A769D, L775A, D801G, K875T,N892Y, N1008D S100D, E229S, D458S, K567R, S582K, S635E, N672D, 30 95 7921612 G753E, S754E, A769D, L775A, D801G, K875T, N892Y E229S, S582K,S635E, T649K, I656V, N672D, P752K, G753E, 30 95 624 1619 S757D, A769D,L775A, D801G, A843P, K875T, N892Y E229S, S635E, T649K, I656V, N672D,G753E, S754R, S757D, 30 95 672 1664 A769D, L775A, D801G, A843P, K875T,N892Y T18D, E229S, D458S, T631N, N672D, M728V, G753E, 30 95 792 1700S754E, S757D, A769D, L775A, D801G, K875T, N892Y E229S, D458S, S582K,T631N, S635E, N672D, M728V, 30 95 792 1704 G753E, S754E, S757D, A769D,L775A, D801G, K875T, N892Y A190Q, E229S, K360G, D458S, S582K, N672D,G753E, 30 95 792 1714 S754E, S757D, A769D, L775A, D801G, K875T, N892Y,N1008D S100D, E229S, K360G, D458S, S582K, N672D, G753E, 30 95 792 1741S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y, N1008D E229S,S635E, N672D, P752R, G753E, S754E, A769D, 30 95 792 1745 L775A, D777K,D801G, K875T, N892Y A159P, E229S, D458S, T631N, N672D, M728V, G753E, 3095 792 1783 S754E, S757D, A769D, L775A, D801G, K875T, N892Y E229S,A492L, S635E, T649K, I656V, N672D, G753E, 30 95 792 1826 S757D, A769D,L775A, D801G, K875T, N892Y S100D, A190Q, E229S, K360G, D458S, S582K,N672D, 30 95 792 1844 G753E, S754E, A769D, L775A, D801G, K875T, N892Y,N1008D S100D, E229S, N440K, S582K, T631N, N672D, G753E, 30 95 624 1907S754E, A769D, L775A, D801G, K875T, N892Y, N1008D E229S, I234V, A492L,N672D, G753E, S754E, A769D, 30 95 624 1924 L775A, D777K, D801G, K875T,N892Y A190Q, E229S, I234V, S582K, N672D, G753E, S754E, 30 95 672 1993S757D, A769D, L775A, D801G, K875T, N892Y E229S, D458S, A492L, T631N,N672D, G753E, S754E, 30 95 624 2378 S757D, A769D, L775A, D801G, K875T,N892Y E229S, A624E, S635E, T649K, I656V, N672D, G753E, S754R, 30 95 6242380 S757D, A769D, L775A, D777K, D801G, K875T, N892Y S100D, E229S,K360G, D458S, S582K, N672D, G753E, 30 95 792 3039 S754E, S757D, A769D,L775A, D801G, A843P, K875T, N892Y, N1008D E229S, D458S, K567R, S582K,S635E, T649K, N672D, 30 95 840 674 G753E, S754E, A769D, L775A, D777R,D801G, K875T, N892Y S100D, E229S, K360G, D458S, S582K, T664K, N672D, 3095 840 1023 G753E, S754R, S757D, A769D, L775A, D801G, A843P, K875T,N892Y, N1008D, K1016T S100D, E229S, K360G, D458S, S582K, T664K, N672D,30 95 840 963 G753E, S754E, S757D, A769D, L775A, D801G, A843P, K875T,N892Y, N1008D, K1016T S100D, E229S, K360G, D458S, S582K, N672D, G753E,30 95 840 982 S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y,A911V, N1008D S100D, E229S, K360G, D458S, S582K, N672D, G753E, 30 95 840906 S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y, A911V,N1008D, K1016T S100D, E229S, K360G, D458S, S582K, S635E, N672D, 30 95840 918 G753E, S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y,N1008D S100D, E229S, K360G, D458S, S582K, T664K, N672D, 30 95 840 962G753E, S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y, A911V,N1008D, K1016T S100D, E229S, K360G, D458S, S582K, N672D, G753E, 30 95840 888 S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y, N1008D,K1016T S100D, E229S, K360G, D458S, S582K, T664K, N672D, 30 95 840 1091G753E, S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y, A911V,N1008D, K1016T S100D, E229S, K360G, D458S, S582K, N672D, G753E, 30 95840 796 S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y, N1008D,K1016T S100D, E229S, K360G, D458S, S582K, S635E, T649K, 30 95 840 984N672D, G753E, S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y,N1008D S100D, E229S, K360G, D458S, S582K, N672D, G753E, 30 95 840 867S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y, N1008D S100D,E229S, K360G, D458S, S582K, N672D, G753E, 30 95 840 876 S754E, S757D,A769D, L775A, D801G, A843P, K875T, N892Y E229S, N440K, S582K, A624E,S635E, N672D, G738L, 30 95 840 689 G753E, S754E, S757D, A769D, L775A,D801G, K875T, N892Y E229S, N399K, D458S, A492H, K567R, S582K, S635E, 3095 840 2256 T649K, N672D, G753E, S754E, A769D, L775A, D777R, D801G,K875T, N892Y A190Q, E229S, I234V, T505I, S582K, N672D, G753E, S754E, 3095 840 698 S757D, A769D, L775A, D801G, K875T, N892Y E229S, D458S, A492H,K567R, S582K, S635E, T649K, 30 95 840 492 N672D, G753E, S754E, A769D,L775A, D777R, D801G, K875T, N892Y E229S, N399K, D458S, K567R, S582K,S635E, N672D, 30 95 840 3540 G753E, S754E, A769D, L775A, D777R, D801G,K875T, N892Y E229S, D458S, A492H, K567R, S582K, S635E, N672D, 30 95 8402837 G753E, S754E, A769D, L775A, D777R, D801G, K875T, N892Y E229S,N399K, D458S, A492H, K567R, S582K, S635E, 30 95 840 536 N672D, G753E,S754E, A769D, L775A, D777R, D801G, K875T, N892Y E229S, N399K, D458S,K567R, S582K, S635E, T649K, 30 95 840 2830 N672D, G753E, S754E, A769D,L775A, D777R, D801G, K875T, N892Y A190Q, E229S, I234V, S582K, N672D,G753E, S754E, 30 95 840 874 S757D, A769D, L775A, D801G, K875T, N892YE229S, N440K, S582K, A624E, S635E, N672D, G738L, 30 95 840 768 G753E,S754E, S757D, A769D, L775A, D801G, K875T, N892Y S100D, E229S, K360G,D458S, S582K, N672D, G753E, 30 95 840 883 S754E, S757D, A769D, L775A,D801G, A843P, K875T, N892Y, N1008D E229S, N399K, D458S, A492H, K567R,S582K, S635E, 30 95 840 1836 T649K, N672D, G753E, S754E, A769D, L775A,D777R, D801G, K875T, N892Y

TABLE 10 Half-life of purified variants: Temperature (T) 32° C.,detergent concentration 90% Incubation Half- T Detergent time lifeMutations (° C.) (%) (h) (h) No mutations (Wild-type) 32 90 1 <0.2Q109R, A769T, L775A, K792Y, K875T 32 90 20 12 L775A, K875T, N892Y,A911V, D933M 32 90 20 13 Q109R, L775A, K792Y, K875T 32 90 20 13 L775A,K875T, N892Y 32 90 20 14 L775A, S851F, K875T,N892Y, A911V 32 90 20 14L775A, K875T, N892Y, A911V, A912T, T923H 32 90 20 14 L775A, K875T,N892Y, D933M 32 90 20 15 E229S, S578R, A769D, L775A, K819R 32 90 20 16E229S, K291R, S578R, P752R, G753E 32 90 20 17 E229S, L775A, K875T, N892Y32 90 20 17 E229S, K291R, S578R, G753E 32 90 20 17 Q109R, S578K, D801G,K875T 32 90 20 17 E229S, S578R, L775A, K819R 32 90 20 18 E229S, S578R,D801G, K819R 32 90 20 18 E229S, S578R, K792Y, K819R 32 90 20 18 Q109R,L775A, K792Y, K875T, N892Y 32 90 20 19 E229S, S578R, A769D, K792Y, K819R32 90 20 19 S578K, L775A, K875T, N892Y 32 90 20 20 E229S, S578R, P752R,G753E, N892Y 32 90 20 20 E229S, S578R, S754E, N892Y 32 90 20 20 E229S,S578R, L775A, N892Y 32 90 20 21 E229S, S578R, L775A, K792Y, K819R 32 9020 21 E229S, S578R, G753E, N892Y 32 90 20 21 Q109R, L775A, K875T, N892Y32 90 20 21 E229S, K291R, S578R, P752K, G753E 32 90 20 21 E229S, K291R,S578R, P752R 32 90 20 21 E229S, S578K, K819R 32 90 20 22 E229S, S578R,P779V, K819R 32 90 20 22 E229S, S578R, K819R 32 90 20 23 Q109R, A769T,L775A, K792Y, D801G, K819R, N892Y 32 90 20 23 Q109R, A769T, L775A,D801G, K819R, N892Y 32 90 20 25 E229S, S578R, P752K, G753E, N892Y 32 9020 25 E229S, S578R, A769Q, K819R 32 90 20 25 E229S, S578R, P752K, S754E,N892Y 32 90 20 27 Q109R, L775A, K875T, N892Y 32 90 20 28 Q109R, A769T,L775A, K792Y, K875T, N892Y 32 90 20 31 Q109R, A769T, L775A, K875T, N892Y32 90 20 33 L775A, D801G, K875T, N892Y 32 90 20 36 Q109R, L775A, P779V,K792Y, D801G, K819R, K875T, 32 90 20 37 N892Y Q109R, A769T, L775A,K875T, N892Y 32 90 20 38 Q109R, E229S, A769T, L775A, K875T, N892Y 32 9020 45 Q109R, P752R, G753E, A769T, L775A, K875T, N892Y 32 90 20 55 Q109R,P752K, G753E, S754E, A769T, L775A, K875T, 32 90 20 56 N892Y Q109R,S754E, A769T, L775A, K875T, N892Y 32 90 20 58 Q109R, P752R, G753E,S754E, A769T, L775A, K875T, 32 90 20 58 N892Y Q109R, P752K, G753E,A769T, L775A, K875T, N892Y 32 90 20 60 Q109R, G753E, A769T, L775A,K875T, N892Y 32 90 20 61 Q109R, G753E, S754E, A769T, L775A, K875T, N892Y32 90 20 62 Q109R, N672D, G753E, S754E, A769T, L775A, K875T, 32 90 16795 N892Y E229S, K360R, S578K, P752R, A769D, L775A, K875T, 32 90 167 98N892Y, A911V E229S, K360R, S578R, N672D, P752R, A769D, L775A, 32 90 16798 K875T, N892Y N672D, G753E, L775A, D801G, K875T, N892Y, A911V 32 90167 108 E229S, K360R, S578K, N672D, P752R, A769D, L775A, 32 90 167 116K875T, N892Y N672D, A769D, L775A, D801G, K875T, N892Y 32 90 167 120T631N, N672D, A769D, L775A, D801G, K875T, N892Y 32 90 167 122 N672D,G753E, L775A, D801G, K875T, N892Y, N1008D 32 90 167 123 A190Q, N672D,A769D, L775A, D801G, K875T, N892Y 32 90 167 124 Q109R, G753E, S754E,A769T, L775A, D801G, K875T, 32 90 167 126 N892Y Q89Y, N672D, A769D,L775A, D801G, K875T, N892Y 32 90 167 129 N672D, G753E, L775A, D801G,A843P, K875T, N892Y 32 90 167 131 L46D, E229S, K360R, S578K, N672D,P752R, A769D, 32 90 167 131 L775A, K875T, N892Y E229S, K360R, S578K,N672D, P752R, A769D, L775A, 32 90 167 133 K875T, N892Y E229S, K360R,S578K, N672D, P752R, A769D, L775A, 32 90 167 135 K875T, N892Y, A912TE229S, K360R, S578K, P752K, G753E, S754E, A769D, 32 90 167 136 L775A,K875T, N892Y E229S, S578K, A769D, L775A, K875T, N892Y 32 90 167 138E229S, S578K, P752K, S754E, K875T, N892Y 32 90 167 140 E229S, K567R,S578K, A769D, L775A, K875T, N892Y 32 90 167 141 E229S, K360R, S578K,P752R, A769D, L775A, D801G, 32 90 167 155 K875T, N892Y N672D, G753E,L775A, D801G, K875T, N892Y 32 90 167 160 E229S, S578K, A769D, L775A,P779V, K792Y, K875T, 32 90 167 167 N892Y E229S, K360R, S578K, T631N,N672D, P752R, A769D, 32 90 167 168 L775A, K875T, N892Y N672D, A769D,L775A, D801G, A843P, K875T, N892Y 32 90 167 171 E229S, K360R, S578K,N672D, P752R, A769D, L775A, 32 90 167 175 K875T, N892Y, A932P E229S,A492L, S578K, T631N, G753E 32 90 167 176 E229S, S578K, P752K, G753E,A769D, L775A, K875T, 32 90 167 182 N892Y E229S, K360R, S578K, N672D,P752R, G753E, A769D, 32 90 167 185 L775A, K875T, N892Y N672D, G753E,S754E, A769D, L775A, D801G, K875T, 32 90 167 187 N892Y E229S, S578K,P752K, G753E, S754E, A769D, L775A, 32 90 167 196 K875T, N892Y E229S,K360R, S578K, N672D, P752R, G753E, S754E, 32 90 167 205 A769D, L775A,K875T, N892Y E229S, K360R, S578K, N672D, P752K, G753E, S754E, 32 90 167216 A769D, L775A, K875T, N892Y E229S, S578K, G753E, A769D, L775A, K875T,N892Y 32 90 167 218 E229S, K360R, S578K, N672D, G753E, S754E, A769D, 3290 167 221 L775A, D845E, K875T, N892Y Q109R, E229S, S578K, P752K 32 90167 225 E229S, S578K, G753E, S754E, A769D, L775A, K875T, 32 90 167 234N892Y E229S, K360R, S578K, N672D, P752K, G753E, A769D, 32 90 167 234L775A, K875T, N892Y E229S, S578K, N672D, A769D, L775A, K875T, N892Y 3290 167 239 E229S, S578K, P752R, G753E, S754E, A769D, L775A, 32 90 167278 K875T, N892Y Q109R, E229S, G753E, S754E, A769T, L775A, D801G, 32 90167 308 K875T, N892Y E229S, S578K, A769D, L775A, D801G, K875T, N892Y 3290 167 337 E229S, N672D, G753E, L775A, D801G, K875T, N892Y 32 90 167 367E229N, N672D, A769D, L775A, D801G, K875T, N892Y 32 90 167 >385 E229S,N672D, A769D, L775A, D801G, K875T, N892Y 32 90 167 >385

TABLE 11 Half-life of purified variants: Temperature (T) 35° C.,detergent concentration 90% Incubation Half- T Detergent time lifeMutations (° C.) (%) (h) (h) No mutations (Wild-type) 35 90 1 <0.2E229N, P752R, G753E, S754E, L775A, D801G, K875T, 35 90 71 27 N892YE229N, G753E, L775A, D801G, K875T, N892Y 35 90 71 27 Q109R, E229S,K451R, A769T, L775A, D801G, K875T, 35 90 70 28 N892Y E229S, S578K,G753E, A912T 35 90 70 28 T631N, P752R, G753E, S754E, A769D, L775A,D801G, 35 90 71 30 E845D, K875T, N892Y E229S, K360R, S578K, P752R,S754E, A769D, L775A, 35 90 71 30 K875T, N892Y Q89Y, Q109R, E229S, A769T,L775A, D801G, K875T, 35 90 70 30 N892Y N672D, A769D, L775A, D801G,K875T, N892Y 35 90 70 33 E229N, A769D, L775A, D801G, K875T, N892Y 35 9071 33 Q109R, N672D, G753E, S754E, A769T, L775A, K875T, 35 90 70 33 N892YQ109R, E229S, S578K, P779V 35 90 70 34 N672D, G753E, L775A, D801G,K875T, N892Y, A911V 35 90 70 34 E229S, S578K, G753E, A912T 35 90 70 34S100D, Q109R, N672D, G753E, S754E, A769T, L775A, 35 90 70 35 K875T,N892Y E229S, K360R, S578K, S635E, T649K, P752R, A769D, 35 90 71 38L775A, D801G, K875T, N892Y Q109R, G753E, S754E, A769E, L775A, D801G,K875T, 35 90 71 38 N892Y E229S, K360R, S578K, N672D, P752R, S754E,A769D, 35 90 71 40 L775A, K875T, N892Y Q109R, E229S, S578K, A912T 35 9070 40 E229S, K360R, S578K, P752R, A769D, L775A, D801G, 35 90 71 42K875T, N892Y Q109R, A159P, E229S, N672D, F746L, G753E, S754E, 35 90 16842 A769T, L775A, D801G, A843P, K875T, N892Y E229S, S578K, G753E, P779V,K792Y, D801G, A912T 35 90 70 42 E229S, K360R, S578K, P752R, S754E,A769D, L775A, 35 90 71 44 D801G, K875T, N892Y E229S, T631N, A769D,L775A, D801G, K875T, N892Y 35 90 166 44 E229S, S578K, P752K, G753E,S754E, D801G, A912T 35 90 70 46 E229N, S754E, A769D, L775A, D801G,K875T, N892Y 35 90 71 47 E229N, N672D, P752K, G753E, A769D, L775A,D801G, 35 90 119 48 K875T, N892Y, N991D E229N, P752K, G753E, A769D,L775A, D801G, K875T, 35 90 120 48 N892Y S100D, E229S, K360R, S578K,T631N, P752R, G753E, 35 90 119 50 S754E, A769D, L775A, D801G, K875T,N892Y E229S, K360R, S578K, T631N, P752R, G753E, S754E, 35 90 119 51A769D, L775A, D801G, K875T, N892Y, N991D E229N, T631N, N672D, A769D,L775A, D801G, K875T, 35 90 70 52 N892Y A58L, E229S, K360R, S578K, N672D,G753E, S754E, A769D, 35 90 70 52 L775A, K875T, N892Y E229S, K360R,S578K, N672D, G753E, S754E, A769D, 35 90 70 56 L775A, D845E, K875T,N892Y Q109R, N672D, G753E, S754E, A769E, L775A, D801G, 35 90 71 56K875T, N892Y E229S, K360R, S578K, T631N, N672D, P752R, G753E, 35 90 11957 S754E, A769D, L775A, D801G, K875T, N892Y E229S, S578K, N672D, A769D,L775A, K875T, N892Y 35 90 120 58 L46D, E229S, K360R, S578K, N672D,G753E, S754E, A769D, 35 90 70 58 L775A, A843P, K875T, N892Y E229N,S635E, T649K, I656V, N672D, A769D, L775A, 35 90 166 59 D801G, K875T,N892Y L46D, E229S, K360R, S578K, T631N, N672D, G753E, S754E, 35 90 70 59A769D, L775A, K875T, N892Y E229S, S578K, P752K, G753E, S754E, A912T 3590 166 61 Q109R, E229S, G753E, S754E, A769T, L775A, D801G, 35 90 166 61K875T, N892Y, A911V E229S, K360R, S578K, P752R, G753E, S754E, A769D, 3590 166 62 L775A, D801G, K875T, N892Y Q109R, E229S, S578K, P752R, G753E35 90 166 62 Q109R, E229S, A769T, L775A, D801G, K875T, N892Y 35 90 16662 L46D, E229S, S578K, G753E, S754E, A769D, L775A, D801G, 35 90 167 63K875T, N892Y Q109R, E229S, T631N, G753E, S754E, A769T, L775A, 35 90 16663 D801G, K875T, N892Y Q109R, E229S, D458S, G753E, S754E, A769T, L775A,35 90 166 64 D801G, K875T, N892Y Q109R, E229S, G753E, S754E, A769T,L775A, D801G, 35 90 166 64 K875T, N892Y S100D, E229S, S578K, G753E,A912T 35 90 166 64 E229S, K360R, S578K, P752K, G753E, S754E, A769D, 3590 71 64 L775A, D801G, K875T, N892Y E229N, P752K, G753E, S754E, A769D,L775A, D801G, 35 90 166 64 K875T, N892Y E229N, S635E, T649K, I656V,N672D, G753E, A769D, 35 90 168 64 L775A, D801G, K875T, N892Y, N991DQ109R, E229S, S635T, G753E, S754E, A769T, L775A, 35 90 166 65 D801G,K875T, N892Y E229N, S635E, T649K, I656V, N672D, S757D, A769D, 35 90 16865 L775A, D801G, A843P, K875T, N892Y E229N, N672D, P752R, S754E, A769D,L775A, D801G, 35 90 70 66 K875T, N892Y E229N, K567R, S635E, T649K,I656V, N672D, G753E, 35 90 168 66 A769D, L775A, D801G, K875T, N892YE229S, S578K, G753E, D801G, A912T 35 90 166 66 Q109R, E229S, G753E,S754E, A769T, L775A, D801G, 35 90 166 66 K875T, N892Y, D901A Q109R,A190Q, E229S, G753E, S754E, A769T, L775A, 35 90 166 66 D801G, K875T,N892Y Q109R, E229S, A769T, L775A, D801G, A843P, K875T, 35 90 166 66N892Y E229N, I234V, S635E, T649K, I656V, N672D, A769D, 35 90 120 66L775A, D801G, A843P, K875T, N892Y E229N, S635E, T649K, I656V, N672D,A769D, L775A, 35 90 168 66 D801G, A843P, K875T, N892Y Q109R, E229S,G753E, S754E, A769T, L775A, D801G, 35 90 166 67 K875T, N892Y, N1008DT631N, A769D, L775A, D801G, K875T, N892Y 35 90 166 67 Q109R, E229S,A492L, A769T, L775A, D801G, K875T, 35 90 166 67 N892Y E229N, P752K,G753E, A769D, L775A, D801G, A843P, 35 90 168 68 K875T, N892Y E229S,S578N, N672D, A769D, L775A, D801G, K875T, 35 90 168 68 N892Y 727\,E229S, K360R, S578K, N672D, G753E, S754E, A769D, 35 90 166 69 L775A,D801G, K875T, N892Y E229S, P752K, G753E, A769D, L775A, D801G, K875T, 3590 168 69 N892Y E229N, S635E, T649K, I656V, N672D, S754E, A769D, 35 90166 69 L775A, D801G, K875T, N892Y L46D, E229S, I234V, K360R, S578K,N672D, G753E, S754E, 35 90 168 69 A769D, L775A, D801G, K875T, N892YE229N, I234V, N672D, G753E, S754E, A769D, L775A, 35 90 168 70 D801G,K875T, N892Y, N1008D L46D, E229S, K360R, S578K, N672D, G753E, S754E,A769D, 35 90 168 70 L775A, D801G, K875T, N892Y E229N, N672D, P752K,A769D, L775A, D801G, K875T, 35 90 166 70 N892Y Q109R, E229S, P752K,G753E, S754E, A769T, L775A, 35 90 166 70 D801G, K875T, N892Y E229N,N672D, A769D, L775A, D801G, K875T, N892Y 35 90 70 70 Q109R, E229S,T631N, S635E, T649K, N672D, P752R, 35 90 167 71 G753E, S754E, A769T,L775A, D801G, K875T, N892Y Q109R, E229S, G753E, S754E, A769D, L775A,P779V, 35 90 166 71 D801G, K875T, N892Y E229N, D458S, N672D, G753E,S754E, A769D, L775A, 35 90 166 72 D801G, K875T, N892Y L46D, E229S,K360R, S578K, N672D, G753E, S754E, A769D, 35 90 168 72 L775A, D801G,K875T, N892Y A58L, E229N, N672D, G753E, S754E, A769D, L775A, 35 90 16673 D801G, K875T, N892Y A159P, E229N, N672D, G753E, S754E, A769D, L775A,35 90 166 73 D801G, K875T, N892Y E229N, S635E, T649K, I656V, N672D,G753E, S754E, 35 90 166 74 A769D, L775A, D801G, K875T, N892Y E229N,N672D, G753E, S754E, A769D, L775A, D801G, 35 90 168 75 K875T, N892Y,N1008D E229S, K360R, S578K, N672D, P752R, A769D, L775A, 35 90 71 75D801G, K875T, N892Y Q109R, A159P, E229S, S635E, G753E, S754E, A769D, 3590 167 75 L775A, D801G, K875T, N892Y E229S, S578K, N672D, G753E, S754E,A769D, L775A, 35 90 168 75 K875T, N892Y E229N, S635E, T649K, I656V,N672D, G753E, A769D, 35 90 168 76 L775A, D801G, K875T, N892Y, T923HE229S, T631N, P752K, G753E, A769D, L775A, D801G, 35 90 167 76 K875T,N892Y E229N, S635E, T649K, I656V, N672D, P752R, G753E, S754E, 35 90 16677 A769D, L775A, D801G, K875T, N892Y E229N, S635E, T649K, I656V, N672D,P752K, G753E, 35 90 168 77 A769D, L775A, D801G, A843P, K875T, N892YA190Q, E229S, K360R, S578K, P752R, G753E, S754E, 35 90 168 77 A769D,L775A, D801G, K875T, N892Y A58L, E229S, I234V, N672D, G753E, S754E,A769D, L775A, 35 90 168 78 D801G, K875T, N892Y L46D, E229S, K360R,S578K, N672D, G753E, S754E, A769D, 35 90 70 78 L775A, K875T, N892Y,A912T L46D, E229S, K360R, S578K, N672D, P752K, G753E, S754E, 35 90 70 78A769D, L775A, K875T, N892Y E229S, N672D, G753E, L775A, D801G, K875T,N892Y, 35 90 166 78 A911V A159P, E229N, N672D, G753E, S754E, A769D,L775A, 35 90 166 79 D801G, K875T, N892Y, N1008D E229N, S635E, N672D,G753E, S754E, A769D, L775A, 35 90 168 80 D801G, K875T, N892Y, N1008DE229N, S635E, T649K, I656V, N672D, A769D, L775A, 35 90 120 80 D801G,A843P, K875T, N892Y, N991D E229N, T631N, N672D, G753E, S754E, A769D,L775A, 35 90 166 80 D801G, K875T, N892Y E229N, N672D, P752K, G753E,S754E, A769D, L775A, 35 90 168 80 D801G, K875T, N892Y, N1008D E229N,N672D, G753E, S754E, A769D, L775A, D801G, 35 90 166 80 K875T, N892Y,V998K Q109R, E229S, K567R, A769T, L775A, D801G, K875T, 35 90 166 81N892Y E229N, S635T, N672D, G753E, S754E, A769D, L775A, 35 90 166 82D801G, K875T, N892Y Q109R, A159P, E229S, S635E, T649K, G753E, S754E, 3590 167 82 A769D, L775A, D801G, K875T, N892Y, N1008D E229N, N672D, G753E,S754E, A769D, L775A, D801G, 35 90 166 82 A843P, K875T, N892Y E229N,N672D, I703L, A769D, L775A, D801G, K875T, 35 90 166 82 N892Y E229S,K360R, S578K, T631N, P752R, G753E, S754E, 35 90 120 82 A769D, L775A,D801G, K875T, N892Y E229N, N672D, I703L, G753E, S754E, A769D, L775A, 3590 166 82 D801G, K875T, N892Y E229S, S635E, T649K, I656V, N672D, A769D,L775A, 35 90 168 83 D801G, K875T, N892Y Q109R, E229S, N672D, P752R,G753E, S754E, A769S, 35 90 168 83 L775A, D801G, K875T, N892Y Q109R,A159P, E229S, L533I, S582K, N672D, M728V, 35 90 168 84 G753E, S754E,A769T, L775A, D801G, A843P, K875T, N892Y Q109R, A159P, E229S, N672D,G753E, S754E, A769T, 35 90 168 84 L775A, D801G, K875T, N892Y, A911VE229N, S635E, T649K, I656V, N672D, P752K, G753E, 35 90 166 84 A769D,L775A, D801G, K875T, N892Y Q109R, A159P, E229S, G753E, S754E, A769T,L775A, 35 90 166 85 D801G, K875T, N892Y E229N, S635E, T649K, I656V,N672D, G753E, A769D, 35 90 168 86 L775A, D801G, K875T, N892Y E229S,K567R, P752K, G753E, A769D, L775A, D801G, 35 90 167 86 K875T, N892YE229N, N672D, G753E, A769D, L775A, D801G, K875T, 35 90 166 87 N892YQ109R, E229S, T631N, G753E, S754E, A769D, L775A, 35 90 166 87 D801G,K875T, N892Y L46D, E229S, K360R, S578K, A624E, N672D, G753E, S754E, 3590 168 87 A769D, L775A, D801G, K875T, N892Y Q109R, A159P, E229S, S635E,T649K, G753E, S754E, 35 90 167 87 A769D, L775A, D801G, K875T, N892Y,A912T E229N, T631N, S635E, T649K, I656V, N672D, A769D, 35 90 70 88L775A, D801G, K875T, N892Y Q109R, E229S, K451R, N672D, G753E, S754E,A769T, 35 90 166 88 L775A, D801G, K875T, N892Y E229N, S635E, T649K,I656V, N672D, P752K, G753E, 35 90 166 89 S754E, A769D, L775A, D801G,K875T, N892Y S100D, Q109R, A159P, E229S, S635E, T649K, G753E, 35 90 16789 S754E, A769D, L775A, D801G, K875T, N892Y E229N, S635E, T649K, I656V,N672D, P752R, G753E, 35 90 168 89 A769D, L775A, D801G, A843P, K875T,N892Y Q109R, E229S, N672D, P752R, G753E, S754E, A769T, 35 90 168 90L775A, D801G, K875T, N892Y, A911V E229N, K567R, S635E, T649K, I656V,N672D, A769D, 35 90 70 90 L775A, D801G, K875T, N892Y Q109R, A159P,E229S, N672D, G753E, S754E, A769T, 35 90 168 90 L775A, D801G, K875T,N892Y E229N, N672D, P752R, G753E, S754E, A769D, L775A, 35 90 166 90D801G, K875T, N892Y E229S, S635E, T649K, I656V, N672D, G753E, S754E, 3590 149 90 A769D, L775A, V800P, D801G, K875T, N892Y Q109R, E229S, S635E,T649K, G753E, S754E, A769D, 35 90 166 91 L775A, D801G, K875T, N892YQ109R, A159P, E229S, G753E, S754E, A769D, L775A, 35 90 168 91 D801G,K875T, N892Y Q109R, E229S, N672D, P752R, G753E, S754E, A769T, 35 90 16891 L775A, D801G, K875T, N892Y, A912T Q109R, A190Q, E229S, G753E, S754E,A769D, L775A, 35 90 166 91 D801G, K875T, N892Y Q109R, A159P, A190Q,E229S, G753E, S754E, A769D, 35 90 168 92 L775A, D801G, K875T, N892YE229S, P752K, G753E, A769D, L775A, D801G, A843P, 35 90 168 92 K875T,N892Y Q109R, A159P, E229S, S635E, T649K, P752R, G753E, S754E, 35 90 16792 A769D, L775A, D801G, K875T, N892Y E229N, N672D, G753E, S754E, A769D,L775A, D801G, 35 90 168 93 K875T, N892Y, N1008D Q109R, E229S, S635T,G753E, S754E, A769D, L775A, 35 90 166 95 D801G, K875T, N892Y Q109R,A159P, E229S, D458S, G753E, S754E, A769D, 35 90 168 95 L775A, D801G,K875T, N892Y E229N, N672D, P752K, G753E, A769D, L775A, D801G, 35 90 16896 A843P, K875T, N892Y E229N, N672D, P752R, G753E, A769D, L775A, D801G,35 90 166 96 K875T, N892Y E229N, N672D, G753E, S754E, A769D, L775A,D801G, 35 90 166 97 K875T, N892Y E229S, S582K, S635E, T649K, I656V,N672D, G738L, 35 90 168 97 G753E, S754E, A769D, L775A, D801G, K875T,N892Y Q109R, A159P, E229S, S635E, T649K, N672D, P752K, 35 90 167 97G753E, S754E, A769T, L775A, D801G, K875T, N892Y Q109R, E229S, T631N,N672D, P752K, G753E, S754E, 35 90 167 98 A769T, L775A, D801G, K875T,N892Y E229N, T631N, N672D, P752K, G753E, A769D, L775A, 35 90 167 98D801G, K875T, N892Y E229N, S635E, T649K, I656V, N672D, P752R, G753E,S754E, 35 90 167 100 A769D, L775A, D801G, A843P, K875T, N892Y E229N,T631N, N672D, G753E, S754E, A769D, L775A, 35 90 168 100 D801G, K875T,N892Y, N1008D E229N, N672D, P752K, G753E, A769D, L775A, D801G, 35 90 167101 K875T, N892Y, T923H Q89Y, Q109R, A159P, E229S, S635E, T649K, N672D,G753E, 35 90 167 101 S754E, A769T, L775A, D801G, K875T, N892Y A190Q,E229S, S635E, T649K, I656V, N672D, I703L, G753E, 35 90 168 102 S754E,A769D, L775A, D801G, K875T, N892Y, N1008D Q109R, E229S, N672D, P752R,G753E, S754E, A769T, 35 90 168 103 L775A, D801G, K875T, N892Y Q109R,E229S, N672D, G753E, S754E, A769T, L775A, 35 90 166 103 D801G, K875T,N892Y Q109R, E229S, G753E, S754E, A769D, L775A, D801G, 35 90 166 103K875T, N892Y Q109R, A159P, E229S, K567R, G753E, S754E, A769D, 35 90 168103 L775A, D801G, K875T, N892Y E229S, D458S, K567R, N672D, G753E, S754E,A769D, 35 90 168 103 L775A, D801G, K875T, N892Y E229S, K360R, S578K,P752R, G753E, S754E, A769D, 35 90 120 104 L775A, D801G, K875T, N892YA159P, E229N, S635E, T649K, I656V, N672D, A769D, 35 90 70 104 L775A,D801G, K875T, N892Y L46D, Q109R, A159P, E229S, N672D, G753E, S754E,A769T, 35 90 168 104 L775A, D801G, A843P, K875T, N892Y Q109R, A159P,E229S, D458S, N672D, G753E, S754E, 35 90 168 104 A769T, L775A, D801G,K875T, N892Y Q109R, A159P, E229S, S635E, T649K, G753E, S754E, 35 90 167104 A769D, L775A, D801G, K875T, N892Y, K1016T Q109R, E229S, N672D,P752R, G753E, S754E, A769T, 35 90 168 107 L775A, D801G, K875T, N892YL46D, A58L, E229S, K360R, S578K, N672D, G753E, S754E, 35 90 70 108A769D, L775A, K875T, N892Y E229S, S635E, T649K, I656V, N672D, G753E,A769D, 35 90 168 109 L775A, D801G, K875T, N892Y Q109R, E229S, N672D,G753E, S754E, A769T, L775A, 35 90 166 109 D801G, K875T, N892Y, N991DQ109R, A159P, E229S, S635E, T649K, N672D, G753E, 35 90 168 109 S754E,A769T, L775A, D801G, K875T, N892Y Q109R, A159P, E229S, S635E, T649K,G753E, S754E, 35 90 168 110 A769D, L775A, D801G, K875T, N892Y Q109R,E229S, N672D, G753E, S754E, A769T, L775A, 35 90 166 110 D801G, K875T,N892Y, T923H L46D, E229S, P752K, G753E, S757D, A769D, L775A, 35 90 149110 D801G, K875T, N892Y A190Q, E229N, N672D, G753E, S754E, A769D, L775A,35 90 166 112 D801G, K875T, N892Y E229S, S635E, T649K, I656V, N672D,S754E, A769D, 35 90 168 112 L775A, D801G, K875T, N892Y Q109R, E229S,G753E, S754E, A769D, L775A, D801G, 35 90 166 112 K875T, N892Y, N1008DE229N, N672D, I703L, P752K, G753E, A769D, L775A, 35 90 167 113 D801G,K875T, N892Y E229S, T631N, S635E, T649K, I656V, N672D, I703L, G753E, 3590 168 113 S754E, A769D, L775A, D801G, K875T, N892Y, N1008D E229S,S635E, T649K, I656V, N672D, I703L, M728V, G753E, 35 90 168 114 S754E,A769D, L775A, D801G, K875T, N892Y, N1008D Q109R, A159P, E229S, G753E,S754E, A769D, L775A, 35 90 168 114 D801G, K875T, N892Y, A912T E229N,N672D, P752K, G753E, A769D, L775A, D801G, 35 90 168 114 K875T, N892YE229S, S635E, T649K, I656V, N672D, I703L, G753E, S754E, 35 90 168 114A769D, L775A, D801G, K875T, N892Y, N1008D E229S, K360G, N672D, G753E,S754E, A769D, L775A, 35 90 168 114 D801G, K875T, N892Y Q109R, A159P,E229S, K567R, N672D, G753E, S754E, 35 90 168 114 A769T, L775A, D801G,K875T, N892Y Q109R, E229S, N672D, G753E, S754E, A769T, L775A, 35 90 166115 D801G, K875T, N892Y, K1016T L46D, E229S, N672D, G753E, S754E, A769D,L775A, 35 90 168 115 D801G, K875T, N892Y Q109R, A159P, E229S, G753E,S754E, A769D, L775A, 35 90 168 115 D801G, K875T, N892Y, N1008D Q109R,A159P, E229S, N672D, G753E, S754E, A769T, 35 90 168 116 L775A, V800P,D801G, A843P, K875T, N892Y Q109R, A159P, E229S, G753E, S754E, A769D,L775A, 35 90 168 116 D801G, A843P, K875T, N892Y Q109R, E229S, G753E,S754E, A769D, L775A, D801G, 35 90 166 117 K875T, N892Y Q109R, A159P,E229S, I703L, G753E, S754E, A769D, L775A, 35 90 168 117 D801G, K875T,N892Y Q109R, E229S, S582K, N672D, G753E, S754E, A769T, 35 90 149 117L775A, D801G, A843P, K875T, N892Y Q109R, E229S, S635E, T649K, I656V,N672D, I703L, G753E, 35 90 168 117 S754E, A769D, L775A, D801G, K875T,N892Y, N1008D Q109R, A159P, E229S, S635E, T649K, N672D, G753E, 35 90 167117 S754E, A769T, L775A, D801G, K875T, N892Y E229S, S635E, T649K, I656V,N672D, P752K, G753E, 35 90 168 117 A769D, L775A, D801G, K875T, N892Y,A912T Q109R, E229S, K567R, T631N, N672D, P752R, G753E, 35 90 149 117S754E, A769D, L775A, D801G, K875T, N892Y Q109R, A190Q, E229S, N672D,G753E, S754E, A769T, 35 90 166 118 L775A, D801G, K875T, N892Y Q109R,A159P, E229S, T631N, G753E, S754E, A769D, 35 90 168 118 L775A, D801G,K875T, N892Y E229S, N440K, S582K, A624E, N672D, G753E, S754E, 35 90 168120 A769D, L775A, D801G, K875T, N892Y Q109R, E229S, N672D, I703L, P752R,G753E, S754E, A769T, 35 90 168 120 L775A, D801G, K875T, N892Y A190Q,E229S, D458S, N672D, G753E, S754E, A769D, 35 90 168 120 L775A, D801G,K875T, N892Y E229S, S635E, T649K, I656V, N672D, G753E, A769D, 35 90 168121 L775A, D801G, K875T, N892Y Q109R, E229S, A624E, N672D, G753E, S754E,A769T, 35 90 149 121 L775A, D801G, A843P, K875T, N892Y E229S, S635E,T649K, I656V, N672D, P752R, G753E, S754E, 35 90 168 121 A769D, L775A,D801G, K875T, N892Y E229S, S635E, T649K, I656V, N672D, G753E, A769D, 3590 168 122 L775A, D801G, K875T, N892Y, K1016T Q109R, E229S, N672D,G753E, S754E, A769T, L775A, 35 90 166 122 D801G, K875T, N892Y, V998KE229N, S635E, T649K, I656V, N672D, P752R, S754E, 35 90 70 122 A769D,L775A, D801G, K875T, N892Y E229S, D458S, T631N, N672D, G753E, S754E,A769D, 35 90 168 122 L775A, D801G, K875T, N892Y E229S, D458S, S635E,N672D, G753E, S754E, A769D, 35 90 168 126 L775A, D801G, K875T, N892Y,N1008D Q109R, E229S, T631N, N672D, P752R, G753E, S754E, 35 90 168 126A769T, L775A, D801G, K875T, N892Y E229S, K360G, S635E, T649K, I656V,N672D, G753E, 35 90 168 126 A769D, L775A, D801G, K875T, N892Y E229N,T631N, N672D, G753E, S754E, A769D, L775A, 35 90 167 126 D801G, K875T,N892Y, N1008D Q109R, A159P, E229S, S635E, T649K, G753E, S754E, 35 90 167127 A769D, L775A, D801G, A843P, K875T, N892Y A159P, E229S, N672D, G753E,S754E, A769D, L775A, 35 90 168 127 D801G, K875T, N892Y E229S, S635E,T649K, I656V, N672D, S757D, A769D, 35 90 168 128 L775A, D801G, K875T,N892Y E229N, S635E, T649K I656V, N672D, P752R, A769D, 35 90 70 128L775A, D801G, K875T, N892Y A159P, E229S, I234V, N672D, G753E, S754E,A769D, 35 90 168 128 L775A, D801G, K875T, N892Y Q109R, E229S, N672D,P752R, G753E, S754E, A769T, 35 90 168 129 L775A, D801G, K875T, N892Y,D901A E229S, S635E, T649K, I656V, N672D, G753E, A769D, 35 90 168 130L775A, V800P, D801G, K875T, N892Y L46D, E229S, K360G, S578K, N672D,G753E, S754E, 35 90 168 130 A769D, L775A, D801G, K875T, N892Y Q109R,A159P, E229S, T631E, S635E, T649K, P752R, 35 90 149 133 G753E, S754E,A769D, L775A, D801G, K875T, N892Y E229S, N672D, G753E, S754E, A769D,L775A, D801G, 35 90 168 135 K875T, N892Y E229S, I234V, N672D, G753E,S754E, A769D, L775A, 35 90 168 135 D801G, K875T, N892Y L46D, E229S,S635E, T649K, I656V, N672D, G753E, S754E, 35 90 149 136 A769D, L775A,D801G, K875T, N892Y Q109R, E229S, T631N, N672D, P752R, G753E, S754E, 3590 167 136 A769D, L775A, D801G, K875T, N892Y S100D, A190Q, E229S, I234V,N672D, G753E, S754E, 35 90 168 137 A769D, L775A, D801G, K875T, N892YE229S, S635E, T649K, I656V, N672D, G753E, S754E, 35 90 168 138 A769D,L775A, D801G, K875T, N892Y E229N, N672D, P752R, G753E, A769D, L775A,D801G, 35 90 166 138 K875T, N892Y E229S, P752K, G753E, S757D, A769D,L775A, D801G, 35 90 167 139 K875T, N892Y E229S, A624E, N672D, G753E,S754E, A769D, L775A, 35 90 168 139 D801G, K875T, N892Y E229S, S635E,T649K, I656V, N672D, I703L, G753E, S754E, 35 90 168 139 A769D, L775A,D801G, K875T, N892Y, N1008D Q109R, E229S, D458S, N672D, G753E, S754E,A769T, 35 90 166 140 L775A, D801G, K875T, N892Y A190Q, E229S, I234V,N672D, G753E, S754E, A769D, 35 90 168 140 L775A, D801G, K875T, N892YE229S, S635E, T649K, I656V, N672D, P752R, G753E, S754E, 35 90 167 141A769D, L775A, D801G, K875T, N892Y Q109R, A159P, E229S, S635E, T649K,N672D, G753E, 35 90 167 141 S754E, S757D, A769T, L775A, D801G, K875T,N892Y Q109R, E229S, N672D, I703L, G753E, S754E, A769T, 35 90 166 141L775A, D801G, K875T, N892Y Q109R, E229S, T631N, N672D, G753E, S754E,A769T, 35 90 166 141 L775A, D801G, K875T, N892Y E229S, T631E, P752K,G753E, S757D, A769D, L775A, 35 90 168 141 D801G, K875T, N892Y E229S,N440K, S582K, N672D, G753E, S754E, S757D, 35 90 168 141 A769D, L775A,D801G, K875T, N892Y, N1008D E229S, K567R, S635E, T649K, I656V, N672D,G753E, 35 90 168 142 A769D, L775A, D801G, K875T, N892Y E229S, S635E,T649K, I656V, N672D, P752K, G753E, 35 90 168 142 A769D, L775A, D801G,K875T, N892Y, T923H, N1008D Q109R, A159P, E229S, N672D, G753E, S754E,A769T, 35 90 168 142 L775A, D801G, K875T, N892Y E229S, A624E, S635E,T649K, I656V, N672D, I703L, G753E, 35 90 168 143 S754E, A769D, L775A,D801G, K875T, N892Y, N1008D A159P, E229S, S635E, T649K, I656V, N672D,G753E, 35 90 168 145 A769D, L775A, D801G, K875T, N892Y E229S, S635E,T649K, I656V, N672D, P752K, G753E, S754E, 35 90 168 145 A769D, L775A,D801G, K875T, N892Y Q109R, E229S, A624E, T631N, N672D, P752R, G753E, 3590 149 146 S754E, A769D, L775A, D801G, K875T, N892Y Q109R, A159P, E229S,S635E, T649K, N672D, G753E, 35 90 168 146 S754E, S757D, A769T, L775A,D801G, K875T, N892Y, N1008D E229S, S582K, S635E, T649K, I656V, N672D,G753E, 35 90 168 146 A769D, L775A, D801G, K875T, N892Y E229S, I234V,V352I, N672D, G753E, S754E, A769D, L775A, 35 90 168 147 D801G, K875T,N892Y T18D, E229S, N672D, G753E, S754E, A769D, L775A, 35 90 168 149D801G, K875T, N892Y, N1008D A159P, E229S, I234V, N672D, G753E, S754E,A769D, 35 90 168 149 L775A, D801G, K875T, N892Y, N1008D E229S, D458S,T631N, N672D, G753E, S754E, A769D, 35 90 168 149 L775A, D801G, K875T,N892Y Q109R, E229S, T631N, N672D, P752R, G753E, S754E, 35 90 149 150A769D, L775A, D801G, K875T, N892Y, T923H E229S, S635E, T649K, I656V,N672D, I703L, G753E, S754E, 35 90 168 150 A769D, L775A, D801G, K875T,N892Y Q109R, E229S, N672D, G753E, S754E, A769E, L775A, 35 90 166 152D801G, K875T, N892Y Q109R, A159P, E229S, N672D, P752R, G753E, S754E, 3590 168 152 A769T, L775A, D801G, K875T, N892Y E229S, N672D, P752K, G753E,A769D, L775A, D801G, 35 90 167 154 K875T, N892Y E229S, S635E, T649K,I656V, N672D, G753E, A769D, 35 90 168 155 L775A, D801G, K875T, N892Y,A911V E229S, D458S, K567R, T631E, N672D, G753E, S754E, 35 90 168 155A769D, L775A, D801G, K875T, N892Y E229S, S635E, T649K, I656V, N672D,I703L, G753E, S754E, 35 90 168 158 A769D, L775A, D801G, K875T, N892Y,N1008D E229S, N440K, S582K, N672D, G753E, S754E, A769D, 35 90 168 158L775A, D801G, K875T, N892Y, N1008D Q109R, A159P, E229S, N672D, G753E,S754E, A769T, 35 90 168 159 L775A, D801G, A843P, K875T, N892Y E229S,S635E, T649K, I656V, N672D, G753E, S754E, 35 90 168 160 A769D, L775A,D801G, K875T, N892Y, K1016T E229S, S635E, N672D, G753E, S754E, A769D,L775A, 35 90 168 160 D801G, K875T, N892Y E229S, N672D, P752R, G753E,S754E, A769D, L775A, 35 90 167 160 D801G, K875T, N892Y E229S, N672D,G753E, S754E, A769D, L775A, D801G, 35 90 168 161 K875T, N892Y E229S,D458S, N672D, G753E, S754E, A769D, L775A, 35 90 149 162 V800P, D801G,K875T, N892Y, N1008D E229S, I234V, N672D, G753E, S754E, A769D, L775A, 3590 168 162 D801G, K875T, N892Y E229S, N672D, G753E, S754E, A769D, L775A,D801G, 35 90 168 166 K875T, N892Y E229N, S635E, T649K, I656V, N672D,A769D, L775A, 35 90 70 166 D801G, K875T, N892Y, N991D Q109R, A159P,E229S, N672D, P752K, G753E, S754E, 35 90 168 167 A769T, L775A, D801G,K875T, N892Y E229S, S635E, T649K, N672D, G753E, S754E, A769D, 35 90 168167 L775A, D801G, K875T, N892Y, N1008D E229S, S635E, T649K, I656V,N672D, P752K, G753E, S757D, 35 90 168 167 A769D, L775A, D801G, K875T,N892Y, N1008D Q109R, E229S, N672D, G753E, S754E, A769T, L775A, 35 90 166167 D801G, A843P, K875T, N892Y E229S, V352I, S635E, T649K, I656V, N672D,I703L, G753E, 35 90 168 168 A769D, L775A, D801G, K875T, N892Y, N1008DQ109R, A159P, E229S, S635E, T649K, N672D, G738L, 35 90 168 168 G753E,S754E, S757D, A769T, L775A, D801G, K875T, N892Y E229S, N440K, S582K,A624E, N672D, G753E, S754E, 35 90 168 169 A769D, L775A, D801G, K875T,N892Y T18D, A159P, E229S, I234V, N672D, G753E, S754E, A769D, 35 90 168173 L775A, D801G, K875T, N892Y E229S, D458S, N672D, G753E, S754E, A769D,L775A, 35 90 168 174 D801G, K875T, N892Y A190Q, E229S, P752K, G753E,S757D, A769D, L775A, 35 90 168 175 D801G, K875T, N892Y E229S, N440K,S582K, A624E, T631N, N672D, G753E, 35 90 168 175 S754E, A769D, L775A,D801G, K875T, N892Y E229S, D458S, A492L, T631N, N672D, G753E, S754E, 3590 168 176 A769D, L775A, D801G, K875T, N892Y E229S, N672D, G753E, S754E,A769D, L775A, D801G, 35 90 168 176 K875T, N892Y Q109R, A159P, E229S,A624E, A626G, S635E, T649K, 35 90 168 176 N672D, G753E, S754E, S757D,A769T, L775A, D801G, K875T, N892Y E229S, N440K, S582K, N672D, P752R,G753E, S754E, 35 90 168 178 A769D, L775A, D801G, K875T, N892Y, N1008DE229S, D458S, T631N, S635E, T649K, N672D, G753E, 35 90 168 178 S754E,A769D, L775A, D801G, K875T, N892Y E229S, S582K, N672D, G753E, S754E,A769D, L775A, 35 90 168 179 D801G, K875T, N892Y E229S, I234V, A492L,N672D, G753E, S754E, A769D, 35 90 168 181 L775A, D801G, K875T, N892YQ109R, E229S, T631N, N672D, P752R, G753E, S754E, 35 90 167 182 S757D,A769T, L775A, D801G, K875T, N892Y E229S, D458S, S582K, N672D, G753E,S754E, A769D, 35 90 168 183 L775A, D801G, K875T, N892Y, N1008D A159P,E229S, S635E, T649K, I656V, N672D, I703L, G753E, 35 90 168 183 S754E,A769D, L775A, D801G, K875T, N892Y, N1008D Q109R, E229S, T631N, N672D,P752R, G753E, S754E, 35 90 168 188 A769D, L775A, D801G, A843P, K875T,N892Y Q109R, E229S, T631N, N672D, P752R, G753E, S754E, 35 90 149 188S757D, A769D, L775A, D801G, K875T, N892Y Q109R, A159P, E229S, S635E,T649K, N672D, I703L, G753E, 35 90 149 188 S754E, S757D, A769T, L775A,D801G, K875T, N892Y Q89Y, E229S, N672D, G753E, S754E, A769D, L775A, 3590 168 188 D801G, K875T, N892Y E229S, S635E, T649K, I656V, N672D, P752K,G753E, 35 90 168 188 A769D, L775A, D801G, A843P, K875T, N892Y E229S,A492L, S635E, T649K, I656V, N672D, G753E, 35 90 168 189 A769D, L775A,D801G, K875T, N892Y E229S, D458S, S582K, A624E, N672D, G753E, S754E, 3590 168 189 A769D, L775A, D801G, K875T, N892Y E229S, D458S, T631N, S635E,N672D, G753E, S754E, 35 90 168 189 A769D, L775A, D801G, K875T, N892YE229S, S635E, T649K, I656V, N672D, P752K, G753E, 35 90 168 193 A769D,L775A, V800P, D801G, K875T, N892Y, N1008D E229S, D458S, N672D, G753E,S754E, A769D, L775A, 35 90 149 193 D801G, K875T, N892Y, N1008D T18D,E229S, N440K, N672D, G753E, S754E, A769D, 35 90 168 194 L775A, D801G,K875T, N892Y A159P, E229S, N440K, N672D, G753E, S754E, A769D, 35 90 168196 L775A, D801G, K875T, N892Y Q109R, A159P, E229S, S635E, T649K, N672D,P752R, 35 90 168 197 G753E, S754E, A769D, L775A, D801G, K875T, N892YE229S, I234V, S635E, T649K, I656V, N672D, G753E, A769D, 35 90 168 197L775A, D801G, K875T, N892Y Q109R, E229S, T631N, N672D, P752R, G753E,S754E, 35 90 149 197 A769D, L775A, D801G, K875T, N892Y, N1008D E229S,D458S, T631E, N672D, G753E, S754E, A769D, 35 90 168 200 L775A, D801G,K875T, N892Y A190Q, E229S, S635E, T649K, I656V, N672D, I703L, G753E, 3590 168 200 S754E, A769D, L775A, V800P, D801G, K875T, N892Y, N1008DE229S, D458S, N672D, P752R, G753E, S754E, A769D, 35 90 168 200 L775A,D801G, K875T, N892Y A190Q, E229S, T631E, S635E, T649K, I656V, N672D,I703L, 35 90 168 203 G753E, S754E, A769D, L775A, D801G, K875T, N892Y,N1008D E229S, N440K, A492L, S582K, A624E, N672D, G753E, 35 90 168 203S754E, A769D, L775A, D801G, K875T, N892Y E229S, D458S, K567R, T631N,N672D, G753E, S754E, 35 90 168 203 A769D, L775A, D801G, K875T, N892YL46D, E229S, K360R, S578K, N672D, G753E, S754E, A769D, 35 90 168 203L775A, D801G, K875T, N892Y E229S, S582K, S635E, T649K, I656V, N672D,I703L, G753E, 35 90 168 204 S754E, A769D, L775A, D801G, K875T, N892Y,N1008D Q109R, A159P, E229S, S635Q, T649K, G753E, S754E, 35 90 167 204S757D, A769D, L775A, D801G, K875T, N892Y T18D, E229S, S635E, T649K,I656V, N672D, G753E, S754E, 35 90 149 205 A769D, L775A, D801G, K875T,N892Y Q109R, A159P, E229S, G753E, S754E, S757D, A769D, 35 90 168 206L775A, D801G, K875T, N892Y E229S, N672D, G753E, S754E, A769D, L775A,D801G, 35 90 168 206 K875T, N892Y E229N, T631N, N672D, P752K, G753E,A769D, L775A, 35 90 149 207 D801G, K875T, N892Y, A911V Q109R, E229S,T631N, S635E, T649K, N672D, P752R, 35 90 149 209 G753E, S754E, A769D,L775A, D801G, K875T, N892Y E229S, D458S, K567R, N672D, M728V, G753E,S754E, 35 90 168 209 A769D, L775A, D801G, K875T, N892Y E229S, I234V,S582K, N672D, G753E, S754E, A769D, 35 90 168 212 L775A, D801G, K875T,N892Y E229S, N440K, S582K, T631N, N672D, G753E, S754E, 35 90 168 213A769D, L775A, D801G, K875T, N892Y, N1008D A190Q, E229S, I234V, S582K,N672D, G753E, S754E, 35 90 168 215 A769D, L775A, D801G, K875T, N892YA190Q, E229S, I234V, A624E, N672D, G753E, S754E, 35 90 168 216 A769D,L775A, D801G, K875T, N892Y E229S, N440K, S582K, A624E, S635E, N672D,G753E, 35 90 168 216 S754E, A769D, L775A, D801G, K875T, N892Y E229S,S635E, T649K, I656V, N672D, G753E, S754E, 35 90 168 218 A769D, L775A,D801G, K875T, N892Y S100D, E229S, S635E, T649K, I656V, N672D, I703L,G753E, 35 90 168 219 A769D, L775A, D801G, A843P, K875T, N892Y, N1008DA190Q, E229S, S635E, T649K, I656V, N672D, I703L, P752R, 35 90 168 224G753E, S754E, A769D, L775A, D801G, K875T, N892Y, N1008D Q89Y, E229S,N440K, S582K, A624E, N672D, G753E, 35 90 168 225 S754E, A769D, L775A,D801G, K875T, N892Y A190Q, E229S, S635E, T649K, I656V, N672D, I703L,G753E, 35 90 168 231 S754E, S757D, A769D, L775A, D801G, K875T, N892Y,N1008D E229S, I234V, N672D, G753E, S754E, A769D, L775A, 35 90 149 235D801G, A843P, K875T, N892Y E229S, S635E, T649K, I656V, N672D, G753E,S754E, S757D, 35 90 168 236 A769D, L775A, D801G, K875T, N892Y T18D,E229S, S582K, N672D, G753E, S754E, A769D, L775A, 35 90 168 239 D801G,K875T, N892Y E229S, D458S, T631N, N672D, G753E, S754E, S757D, 35 90 168239 A769D, L775A, D801G, K875T, N892Y E229S, S635E, T649K, I656V, N672D,P752R, G753E, S754E, 35 90 168 239 A769D, L775A, D801G, K875T, N892Y,N1008D E229S, N440K, S582K, A624E, N672D, G753E, S754E, 35 90 168 242A769D, L775A, V800P, D801G, K875T, N892Y A190Q, E229S, D458S, T631N,N672D, G753E, S754E, 35 90 168 245 A769D, L775A, D801G, K875T, N892YE229S, K360G, D458S, S582K, N672D, G753E, S754E, 35 90 168 251 A769D,L775A, D801G, K875T, N892Y, N1008D S100D, E229S, S635E, T649K, I656V,N672D, G753E, S754E, 35 90 149 253 A769D, L775A, D801G, K875T, N892YA190Q, E229S, S635E, T649K, I656V, N672D, G753E, 35 90 149 261 S754E,A769D, L775A, D801G, K875T, N892Y S100D, A190Q, E229S, S635E, T649K,I656V, N672D, I703L, 35 90 168 263 G753E, S754E, A769D, L775A, D801G,K875T, N892Y, N1008D E229S, D458S, K567R, S635E, N672D, G753E, S754E, 3590 168 266 A769D, L775A, D801G, K875T, N892Y E229S, D458S, N672D, G753E,S754E, A769D, L775A, 35 90 149 281 D801G, A843P, K875T, N892Y E229S,T631N, N672D, I703L, P752K, G753E, A769D, 35 90 149 284 L775A, D801G,K875T, N892Y E229S, N440K, S582K, N672D, G753E, S754E, A769D, 35 90 149296 L775A, D801G, A843P, K875T, N892Y, N1008D

TABLE 12 Half-life of purified variants: Temperature (T) 35° C.,detergent concentration 95% Incubation Half- T Detergent time lifeMutations (° C.) (%) (h) (h) No mutations (Wild-type) 35 95 1 <0.2E229S, K360G, D458S, S582K, T631N, S635E, N672D, I703L, 35 95 168 40M728V, G753E, S754E, S757D, A769D, L775A, D801G, K875T, N892Y E229S,D458S, S582K, T631N, S635E, N672D, M728V, 35 95 168 27 G753E, S754E,S757D, A769D, L775A, D777R, V800P, D801G, K875T, N892Y E229S, D458S,S582K, T631N, S635E, N672D, M728V, 35 95 168 39 G753E, S754E, S757D,A769D, L775A, D801G, A843P, K875T, N892Y E229S, D458S, S582K, T631N,S635E, N672D, M728V, 35 95 168 46 G753E, S754E, S757D, A769D, L775A,D801G, A843P, K875T, N892Y E229S, D458S, S582K, T631N, S635E, T664K,N672D, 35 95 168 41 M728V, G753E, S754E, S757D, A769D, L775A, D801G,K875T, N892Y E229S, V352I, D458S, S582K, T631N, S635E, N672D, 35 95 16843 M728V, G753E, S754E, S757D, A769D, L775A, D801G, K875T, N892Y, K1016TS100D, E229S, K360G, A624E, S635E, T649K, N672D, 35 95 168 54 G753E,S754E, A769D, L775A, D777K, D801G, K875T, N892Y, K1016T S100D, E229S,V352I, K360G, D458S, A624E, S635E, T649K, 35 95 168 42 I656V, N672D,G753E, S754R, S757D, A769D, L775A, D777K, D801G, K875T, N892Y, K1016TE229S, A624E, S635E, T649K, I656V, N672D, L748T, G753E, 35 95 168 37S754R, S757D, A769D, L775A, D777K, D801G, K875T, N892Y E229S, S582K,A624E, S635E, T649K, I656V, N672D, 35 95 168 38 G753E, S754R, S757D,A769D, L775A, D777K, V800P, D801G, K875T, N892Y E229S, A624E, S635E,T649K, N672D, G753E, S754E, 35 95 168 45 A769D, L775A, D777K, D801G,K875T, N892Y E229S, A624E, S635E, T649K, I656V, N672D, G753E, S754R, 3595 168 55 S757D, A769D, L775A, D777R, D801G, A843P, K875T, N892Y E229S,D458S, K567R, S582K, S635E, N672D, M728V, 35 95 168 23 G753E, S754R,S757D, A769D, L775A, D777K, D801G, K875T, N892Y, K1016T E229S, D458S,K567R, S582K, S635E, N672D, G753E, 35 95 168 26 S754E, A769D, L775A,D777R, V800P, D801G, K875T, N892Y E229S, D458S, K567R, S582K, S635E,N672D, G753E, 35 95 168 29 S754E, A769D, L775A, D777R, D801G, K875T,N892Y, N1008D E229S, D458S, K567R, S582K, S635E, T649K, N672D, 35 95 16829 G753E, S754E, A769D, L775A, D777K, D801G, K875T, N892Y E229S, N399K,D458S, A492H, K567R, S582K, S635E, 35 95 168 61 T649K, N672D, G753E,S754E, A769D, L775A, D777R, D801G, K875T, N892Y E229S, D458S, K567R,S582K, S635E, N672D, G753E, 35 95 168 23 S754E, S757D, A769D, L775A,D777R, D801G, K875T, N892Y E229S, D458S, K567R, S582K, S635E, T664K,N672D, 35 95 168 25 G753E, S754E, A769D, L775A, D777R, K792Y, D801G,K875T, N892Y E229S, D458S, K567R, S582K, S635E, T664K, N672D, 35 95 16825 G753E, S754E, A769D, L775A, D777R, D801G, K875T, N892Y E229S, D458S,K567R, S582K, S635E, N672D, M728V, 35 95 168 30 G753E, S754E, A769D,L775A, D777R, D801G, K875T, N892Y S100D, E229S, K360G, D458S, S582K,A624E, N672D, 35 95 168 60 G753E, S754E, S757D, A769D, L775A, D801G,A843P, K875T, N892Y, N1008D S100D, E229S, K360G, D458S, S582K, N672D,G753E, 35 95 168 50 S754E, S757D, A769D, L775A, D801G, A843P, K875T,N892Y, N1008D S100D, E229S, K360G, D458S, S582K, S635E, N672D, 35 95 16851 G753E, S754E, S757D, A769D, L775A, V800P, D801G, A843P, K875T, N892Y,N1008D S100D, E229S, K360G, D458S, S582K, S635E, T649K, 35 95 168 59N672D, G753E, S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y,N1008D E229S, S635E, T649R, N672D, P752R, G753E, S754E, 35 95 168 37A769D, L775A, D777K, D801G, K875T, N892Y E229S, N672D, P752R, G753E,S754E, S757D, A769D, 35 95 168 47 L775A, D801G, A843P, K875T, N892YE229S, S635E, T649K, I656V, N672D, G753E, A769D, 35 95 168 24 L775A,D801G, K875T, N892Y S100D, E229S, K360G, D458S, S582K, N672D, G753E, 3595 168 45 S754E, A769D, L775A, D801G, K875T, N892Y, N1008D A190Q, E229S,K360G, D458S, S582K, N672D, G753E, 35 95 168 41 S754E, A769D, L775A,D801G, K875T, N892Y, N1008D

TABLE 13 Half-life of purified variants: Temperature (T) 37° C.,detergent concentration 90% Incubation Half- T Detergent time lifeMutations (° C.) (%) (h) (h) No mutations (Wild-type) 37 90 1 <0.2Q109R, A159P, E229S, V352I, S635E, T649K, N672D, I703L, 37 90 120 29G753E, S754E, S757D, A769T, L775A, D801G, K875T, N892Y E229N, T631N,N672D, G753E, S754E, A769D, L775A, 37 90 120 40 D801G, K875T, N892Y,N1008D E229S, N440K, S582K, A624E, S635E, N672D, G753E, 37 90 120 53S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y E229S, I234V,T631N, N672D, I703L, P752K, G753E, A769D, 37 90 120 63 L775A, D801G,K875T, N892Y Q109R, A159P, E229S, S582K, S635Q, T649K, G753E, 37 90 12064 S754E, S757D, A769D, L775A, D801G, K875T, N892Y Q109R, A159P, A190Q,E229S, G753E, S754E, S757D, 37 90 120 65 A769D, L775A, D801G, K875T,N892Y A159P, E229S, S635E, T649K, I656V, N672D, G753E, S754E, 37 90 12067 A769D, L775A, V800P, D801G, K875T, N892Y T18D, Q89Y, E229S, S635E,T649K, I656V, N672D, G753E, 37 90 120 69 S754E, A769D, L775A, D801G,K875T, N892Y T18D, A190Q, E229S, S635E, T649K, I656V, N672D, G753E, 3790 120 70 S754E, A769D, L775A, D801G, K875T, N892Y Q109R, A159P, E229S,T631N, G753E, S754E, S757D, 37 90 120 71 A769D, L775A, D801G, K875T,N892Y A58L, E229S, N440K, S582K, T631N, N672D, G753E, S754E, 37 90 12071 A769D, L775A, D801G, K875T, N892Y, N1008D E229S, S635E, T649K, I656V,N672D, G753E, S754E, 37 90 120 72 A769D, L775A, V800P, D801G, K875T,N892Y E229S, T631N, N672D, I703L, P752R, G753E, A769D, 37 90 120 73L775A, D801G, K875T, N892Y T18D, Q109R, E229S, T631N, S635E, T649K,N672D, P752R, 37 90 120 74 G753E, S754E, A769D, L775A, D801G, K875T,N892Y A190Q, E229S, D458S, K567R, S635E, N672D, G753E, 37 90 120 74S754E, A769D, L775A, D801G, K875T, N892Y Q109R, A159P, E229S, T631E,S635Q, T649K, G753E, 37 90 120 77 S754E, S757D, A769D, L775A, D801G,K875T, N892Y E229S, T631N, N672D, I703L, P752K, G753E, A769D, 37 90 12077 L775A, D801G, K875T, N892Y E229S, T631N, N672D, I703L, P752K, G753E,A769D, 37 90 120 79 L775A, D801G, K875T, N892Y, N1008D Q89Y, E229S,D458S, T631N, N672D, G753E, S754E, S757D, 37 90 120 80 A769D, L775A,D801G, K875T, N892Y A190Q, E229S, D458S, T631N, N672D, G753E, S754E, 3790 120 80 A769D, L775A, D801G, K875T, N892Y Q109R, E229S, T631N, N672D,M728V, P752R, G753E, 37 90 120 82 S754E, S757D, A769D, L775A, D801G,K875T, N892Y Q109R, A159P, E229S, T631N, S635Q, T649K, G753E, 37 90 12082 S754E, S757D, A769D, L775A, D801G, K875T, N892Y Q109R, A159P, E229S,S635Q, T649K, G753E, S754E, 37 90 120 82 S757D, A769D, L775A, D801G,K875T, N892Y Q109R, A159P, E229S, S635E, T649K, N672D, I703L, 37 90 12083 M728V, G753E, S754E, S757D, A769T, L775A, D801G, K875T, N892Y E229S,D458S, T631N, N672D, G753E, S754E, S757D, 37 90 120 83 A769D, L775A,D801G, K875T, N892Y E229S, D458S, S582K, T631N, N672D, M728V, G753E, 3790 120 84 S754E, S757D, A769D, L775A, D801G, K875T, N892Y Q109R, E229S,S582K, T631N, S635E, T649K, N672D, 37 90 120 84 P752R, G753E, S754E,A769D, L775A, D801G, K875T, N892Y A190Q, E229S, D458S, T631N, S635E,N672D, G753E, 37 90 120 84 S754E, A769D, L775A, D801G, K875T, N892YE229S, T631N, N672D, I703L, P752R, G753E, S754E, A769D, 37 90 120 84L775A, D801G, K875T, N892Y E229S, S635E, T649K, I656V, N672D, P752K,G753E, S754E, 37 90 120 84 A769D, L775A, V800P, D801G, K875T, N892YL46D, E229S, T631N, N672D, I703L, P752K, G753E, A769D, 37 90 120 84L775A, D801G, K875T, N892Y A190Q, E229S, R284G, S635E, T649K, I656V,N672D, 37 90 120 85 G753E, S754E, A769D, L775A, V800P, D801G, K875T,N892Y T18D, A190Q, E229S, D458S, T631N, N672D, G753E, S754E, 37 90 12085 A769D, L775A, D801G, K875T, N892Y E229S, S635E, T649K, I656V, N672D,G753E, S754E, 37 90 120 86 A769D, L775A, V800P, D801G, A843P, K875T,N892Y E229S, I234V, N440K, S582K, A624E, S635E, N672D, 37 90 120 86G753E, S754E, A769D, L775A, D801G, K875T, N892Y Q109R, A159P, E229S,S635E, T649K, N672D, I703L, G753E, 37 90 120 86 S754E, S757D, A769T,L775A, V800P, D801G, K875T, N892Y Q109R, E229S, K360G, T631N, S635E,T649K, N672D, 37 90 120 87 P752R, G753E, S754E, A769D, L775A, D801G,K875T, N892Y E229S, S582K, T631N, N672D, I703L, P752K, G753E, 37 90 12087 A769D, L775A, D801G, K875T, N892Y E229S, I234V, S582K, N672D, M728V,G753E, S754E, 37 90 120 87 A769D, L775A, D801G, K875T, N892Y E229S,A492L, S635E, T649K, I656V, N672D, G753E, S754E, 37 90 120 87 A769D,L775A, V800P, D801G, K875T, N892Y E229S, N440K, S582K, A624E, S635E,N672D, G753E, 37 90 120 87 S754E, A769D, L775A, D777K, D801G, K875T,N892Y L46D, E229S, N440K, S582K, T631N, N672D, G753E, S754E, 37 90 12088 A769D, L775A, D801G, K875T, N892Y, N1008D Q109R, A159P, E229S, A624E,S635E, T649K, N672D, I703L, 37 90 120 88 G753E, S754E, S757D, A769T,L775A, D801G, K875T, N892Y E229S, N672D, G753E, S754E, A769D, L775A,D801G, 37 90 120 88 K875T, N892Y E229S, A492L, S635E, T649K, I656V,N672D, G753E, 37 90 120 88 A769D, L775A, D801G, K875T, N892Y, N1008DQ109R, A159P, E229S, T631N, S635E, T649K, N672D, I703L, 37 90 120 88G753E, S754E, S757D, A769T, L775A, D801G, K875T, N892Y T18D, S100D,E229S, S635E, T649K, I656V, N672D, I703L, 37 90 120 88 G753E, A769D,L775A, D801G, A843P, K875T, N892Y, N1008D T18D, E229S, S582K, T664K,N672D, G753E, S754E, A769D, 37 90 120 89 L775A, D801G, K875T, N892YQ109R, A159P, E229S, T631N, S635E, T649K, N672D, 37 90 120 89 P752R,G753E, S754E, A769D, L775A, D801G, K875T, N892Y A58L, E229S, S635E,T649K, I656V, N672D, G753E, S754E, 37 90 120 91 S757D, A769D, L775A,D801G, K875T, N892Y E229S, V352I, D458S, K567R, S635E, N672D, G753E,S754E, 37 90 120 92 A769D, L775A, D801G, K875T, N892Y E229S, S635E,T649K, I656V, N672D, P752R, G753E, S754E, 37 90 120 92 A769D, L775A,D801G, K875T, N892Y, N1008D, K1016T T18D, S100D, E229S, S635E, T649K,I656V, N672D, G753E, 37 90 120 93 S754E, A769D, L775A, D801G, K875T,N892Y T18D, E229S, A492L, S635E, T649K, I656V, N672D, G753E, 37 90 12093 A769D, L775A, D801G, K875T, N892Y E229S, N440K, D458S, S582K, A624E,S635E, N672D, 37 90 120 93 G753E, S754E, A769D, L775A, D801G, K875T,N892Y T18D, E229S, Q372H, S582K, N672D, G753E, S754E, 37 90 120 93A769D, L775A, D801G, K875T, N892Y E229S, D458S, T631N, N672D, G753E,S754E, S757D, 37 90 120 93 A769D, L775A, D801G, K875T, N892Y S100D,E229S, N440K, S582K, A624E, S635E, N672D, 37 90 120 93 G753E, S754E,A769D, L775A, D801G, K875T, N892Y S100D, E229S, S635E, T649K, I656V,N672D, G753E, S754E, 37 90 120 94 A769D, L775A, V800P, D801G, K875T,N892Y E229S, I234V, S582K, N672D, I703L, G753E, S754E, A769D, 37 90 12095 L775A, D801G, K875T, N892Y T18D, E229S, S582K, N672D, G753E, S754E,A769D, L775A, 37 90 120 95 D801G, E856D, K875T, N892Y Q109R, A159P,E229S, S635Q, T649K, G753E, S754E, 37 90 120 95 S757D, A769D, L775A,D801G, A843P, K875T, N892Y E229S, T631N, N672D, I703L, P752K, G753E,A769D, 37 90 120 96 L775A, D801G, K875T, N892Y, K1016T A159P, E229S,A492L, S635E, T649K, I656V, N672D, 37 90 120 96 G753E, A769D, L775A,D801G, K875T, N892Y E229S, T631N, N672D, I703L, P752K, G753E, A769D, 3790 120 96 L775A, D801G, A843P, K875T, N892Y E229S, N440K, S582K, T631N,S635E, T649K, N672D, 37 90 120 96 G753E, S754E, A769D, L775A, D801G,K875T, N892Y, N1008D E229S, D458S, K567R, S582K, S635E, N672D, G753E, 3790 120 96 S754E, A769D, L775A, D777R, D801G, K875T, N892Y T18D, E229S,S582K, N672D, G753E, S754E, A769D, L775A, 37 90 120 97 D801G, K875T,N892Y, I900G L46D, E229S, K360G, D458S, S582K, N672D, G753E, S754E, 3790 120 97 A769D, L775A, D801G, K875T, N892Y, N1008D T18D, E229S, S582K,N672D, G753E, S754E, A769D, L775A, 37 90 120 97 D801G, K819T, K875T,N892Y S100D, E229S, K360G, D458S, S582K, N672D, G753E, 37 90 120 97S754E, A769D, L775A, D777R, V800P, D801G, K875T, N892Y, N1008D E229S,D458S, K567R, S582K, S635E, N672D, G753E, 37 90 120 98 S754E, A769D,L775A, D801G, K875T, N892Y E229S, A624E, S635E, T649K, I656V, N672D,P752R, G753E, 37 90 120 100 S754E, A769D, L775A, D801G, K875T, N892Y,N1008D E229S, N440K, K567R, S582K, A624E, S635E, N672D, 37 90 120 100G753E, S754E, A769D, L775A, D801G, K875T, N892Y T18D, E229S, S582K,N672D, G753E, S754E, P764V, A769D, 37 90 120 100 L775A, D801G, K875T,N892Y A190Q, E229S, A492L, S635E, T649K, I656V, N672D, 37 90 120 100G753E, A769D, L775A, D801G, K875T, N892Y E229S, N440K, S582K, T631E,N672D, G753E, S754E, 37 90 120 100 A769D, L775A, D801G, A843P, K875T,N892Y, N1008D T18D, Q89Y, E229S, S582K, N672D, G753E, S754E, A769D, 3790 120 100 L775A, D801G, K875T, N892Y S100D, Q109R, A159P, E229S, S635E,T649K, N672D, I703L, 37 90 120 102 G753E, S754E, S757D, A769T, L775A,D801G, K875T, N892Y Q109R, A159P, E229S, S635E, T649K, N672D, P752R, 3790 120 102 G753E, S754E, A769D, L775A, D801G, K875T, N892Y T18D, E229S,S582K, N672D, G753E, S754E, A769D, L775A, 37 90 120 102 D801G, P867S,K875T, N892Y E229S, K567R, S635E, T649K, I656V, N672D, P752K, 37 90 120102 G753E, A769D, L775A, D801G, A843P, K875T, N892Y E229S, D458S, S635E,T649K, I656V, N672D, G753E, S754E, 37 90 120 104 A769D, L775A, V800P,D801G, K875T, N892Y E229S, S635E, T649K, I656V, N672D, P752K, G753E,S754E, 37 90 120 104 A769D, L775A, D801G, A843P, K875T, N892Y Q109R,E229S, T631N, S635E, N672D, P752R, G753E, 37 90 120 105 S754E, S757D,A769D, L775A, D801G, K875T, N892Y E229S, A624E, S635E, T649K,I656V,N672D, G753E, S754R, 37 90 120 106 S757D, A769D, L775A, D777R,D801G, K875T, N892Y E229S, T631N, S635E, T649K, I656V, N672D, P752K, 3790 120 106 G753E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y E229S,D458S, S635E, T649K, I656V, N672D, G753E, S754R, 37 90 120 106 S757D,A769D, L775A, D801G, K875T, N892Y E229S, D458S, T631N, N672D, G753E,S754E, S757D, 37 90 120 107 A769D, L775A, D777K, D801G, K875T, N892YE229S, A624E, S635E, T649K, I656V, N672D, G753E, S754R, 37 90 120 107S757D, A769D, L775A, D801G, K875T, N892Y T18D, E229S, S582K, N672D,G753E, S754E, S757D, A769D, 37 90 120 107 L775A, D801G, K875T, N892YE229S, I234V, D458S, S582K, N672D, G753E, S754E, 37 90 120 108 A769D,L775A, D801G, K875T, N892Y Q109R, E229S, K567R, T631N, N672D, P752R,G753E, 37 90 120 109 S754E, S757D, A769D, L775A, D801G, K875T, N892YS100D, A190Q, E229S, D458S, T631N, N672D, G753E, 37 90 120 109 S754E,A769D, L775A, D801G, K875T, N892Y S100D, E229S, S582K, S635E, T649K,I656V, N672D, I703L, 37 90 120 109 G753E, A769D, L775A, D801G, A843P,K875T, N892Y, N1008D E229S, T631N, N672D, I703L, P752K, G753E, S754E, 3790 120 109 A769D, L775A, D801G, K875T, N892Y A190Q, E229S, I234V, S582K,N672D, G753E, S754E, 37 90 120 110 A769D, L775A, D801G, A843P, K875T,N892Y E229S, D458S, T631E, N672D, G753E, S754E, A769D, 37 90 120 111L775A, D777R, D801G, K875T, N892Y T18D, E229S, D458S, K567R, S582K,S635E, N672D, G753E, 37 90 120 111 S754E, A769D, L775A, D801G, K875T,N892Y E229S, N440K, D458S, S582K, T631N, N672D, G753E, 37 90 120 111S754E, A769D, L775A, D801G, K875T, N892Y, N1008D E229S, D458S, K567R,S582K, T631N, S635E, N672D, 37 90 120 112 G753E, S754E, A769D, L775A,D801G, K875T, N892Y E229S, K360G, D458S, S582K, N672D, P752R, G753E, 3790 120 112 S754E, A769D, L775A, D801G, K875T, N892Y, N1008D E229S,D458S, S582K, T631N, N672D, G753E, S754E, 37 90 120 112 S757D, A769D,L775A, D801G, K875T, N892Y E229S, K360G, D458S, S582K, S635E, N672D,G753E, 37 90 120 112 S754E, A769D, L775A, D801G, K875T, N892Y, N1008DE229S, S582K, S635E, T649K, I656V, N672D, P752K, G753E, 37 90 120 112A769D, L775A, D801G, A843P, K875T, N892Y E229S, D458S, K567R, S582K,S635E, N672D, G753E, 37 90 120 112 S754E, A769D, L775A, D801G, K875T,N892Y, K1016T E229S, T631N, N672D, P752R, G753E, S754E, A769D, 37 90 120113 L775A, D801G, A843P, K875T, N892Y E229S, N440K, S582K, N672D, P752R,G753E, S754E, 37 90 120 113 A769D, L775A, D801G, A843P, K875T, N892Y,N1008D S100D, E229S, S635E, T649K, I656V, N672D, I703L, G753E, 37 90 120114 A769D, L775A, D777R, D801G, A843P, K875T, N892Y, N1008D E229S,D458S, K567R, S582K, S635E, N672D, G753E, 37 90 120 115 S754E, A769D,L775A, D777K, D801G, K875T, N892Y E229S, D458S, K567R, T631N, S635E,N672D, G753E, 37 90 120 115 S754E, A769D, L775A, D801G, K875T, N892YA190Q, E229S, D458S, T631N, N672D, G753E, S754E, 37 90 120 115 A769D,L775A, D801G, K875T, N892Y E229S, N440K, S582K, A624E, S635E, N672D,G753E, 37 90 120 115 S754E, S757D, A769D, L775A, D801G, K875T, N892YA190Q, E229S, D458S, T631N, N672D, G753E, S754E, 37 90 120 116 S757D,A769D, L775A, D801G, K875T, N892Y Q109R, A159P, E229S, S635Q, T649K,N672D, G753E, 37 90 120 116 S754E, S757D, A769D, L775A, D801G, K875T,N892Y E229S, N440K, S582K, N672D, P752R, G753E, S754E, 37 90 120 116A769D, L775A, D801G, A843P, K875T, N892Y A190Q, E229S, T631N, N672D,I703L, P752K, G753E, 37 90 120 116 A769D, L775A, D801G, K875T, N892YT18D, E229S, S582K, N672D, G753E, S754E, A769D, L775A, 37 90 120 117D801G, K875T, N892Y E229S, D458S, T631E, S635E, N672D, G753E, S754E, 3790 120 117 A769D, L775A, D801G, K875T, N892Y E229S, A492L, S582K, S635E,T649K, I656V, N672D, 37 90 120 118 G753E, A769D, L775A, D801G, K875T,N892Y S100D, E229S, S635E, T649K, I656V, N672D, P752K, G753E, 37 90 120118 S754E, A769D, L775A, D801G, K875T, N892Y E229S, K360G, D458S, S582K,N672D, G753E, S754E, 37 90 120 118 A769D, L775A, V800P, D801G, K875T,N892Y, N1008D E229S, A624E, S635E, T649K, I656V, N672D, M728V, 37 90 120118 G753E, S754R, S757D, A769D, L775A, D801G, K875T, N892Y E229S, D458S,K567R, S635E, N672D, I703L, G753E, S754E, 37 90 120 118 A769D, L775A,D801G, K875T, N892Y Q109R, E229S, K360G, T631N, N672D, P752R, G753E, 3790 120 119 S754E, S757D, A769D, L775A, D801G, K875T, N892Y E229S, K360G,D458S, K567R, S582K, S635E, N672D, 37 90 120 119 G753E, S754E, A769D,L775A, D801G, K875T, N892Y E229S, N440K, A492L, S582K, T631N, N672D,G753E, 37 90 120 120 S754E, A769D, L775A, D801G, K875T, N892Y, N1008DE229S, I234V, S582K, N672D, G753E, S754E, S757D, 37 90 120 121 A769D,L775A, D801G, K875T, N892Y E229S, N440K, A492L, S582K, A624E, S635E,N672D, 37 90 120 121 G753E, S754E, A769D, L775A, D801G, K875T, N892YA159P, A190Q, E229S, I234V, S582K, N672D, G753E, 37 90 120 122 S754E,A769D, L775A, D801G, K875T, N892Y S100D, E229S, D458S, K567R, S635E,N672D, G753E, 37 90 120 123 S754E, A769D, L775A, D801G, K875T, N892YA190Q, E229S, N440K, S582K, T631N, N672D, G753E, 37 90 120 124 S754E,A769D, L775A, D801G, K875T, N892Y, N1008D S100D, Q109R, E229S, R284G,T631N, S635E, T649K, 37 90 120 125 N672D, P752R, G753E, S754E, A769D,L775A, D801G, K875T, N892Y E229S, V352I, S635E, T649K, I656V, N672D,G753E, S754E, 37 90 120 125 A769D, L775A, V800P, D801G, K875T, N892YT18D, Q109R, E229S, T631N, N672D, P752R, G753E, S754E, 37 90 120 126S757D, A769D, L775A, D801G, K875T, N892Y S100D, A190Q, E229S, I234V,S582K, N672D, G753E, 37 90 120 129 S754E, A769D, L775A, D801G, K875T,N892Y S100D, E229S, K360G, D458S, S582K, N672D, G753E, 37 90 120 129S754E, A769D, L775A, D777K, D801G, K875T, N892Y, N1008D E229S, K360G,D458S, A492L, S582K, N672D, G753E, 37 90 120 130 S754E, A769D, L775A,D801G, K875T, N892Y, N1008D E229S, K360G, D458S, S582K, N672D, G753E,S754E, 37 90 120 130 A769D, L775A, D801G, K875T, N892Y, N1008D S100D,A190Q, E229S, K360G, D458S, S582K, N672D, 37 90 120 132 G753E, S754E,A769D, L775A, D801G, K875T, N892Y, N1008D E229S, A492L, S635E, T649K,I656V, N672D, G753E, 37 90 120 134 S757D, A769D, L775A, D801G, K875T,N892Y E229S, A624E, S635E, T649K, I656V, N672D, G753E, S754R, 37 90 120135 S757D, A769D, L775A, D777K, D801G, K875T, N892Y A190Q, E229S, S635E,T649K, I656V, N672D, P752K, 37 90 120 135 G753E, A769D, L775A, D801G,A843P, K875T, N892Y T18D, E229S, A624E, S635E, T649K, I656V, N672D,G753E, 37 90 120 136 S754R, S757D, A769D, L775A, D777K, D801G, K875T,N892Y E229S, S582K, S635E, N672D, P752R, G753E, S754E, 37 90 120 136A769D, L775A, D801G, K875T, N892Y, N1008D A190Q, E229S, N440K, S582K,A624E, S635E, N672D, 37 90 120 136 G753E, S754E, A769D, L775A, D801G,K875T, N892Y E229S, S582K, S635E, T649K, I656V, N672D, M728V, 37 90 120137 G753E, S754R, S757D, A769D, L775A, D801G, K875T, N892Y E229S, D458S,K567R, S582K, S635E, N672D, I703L, G753E, 37 90 120 137 S754E, A769D,L775A, D801G, K875T, N892Y E229S, I234V, S582K, N672D, G753E, S754E,A769D, 37 90 120 137 L775A, V800P, D801G, K875T, N892Y E229S, S635E,T649K, I656V, N672D, P752K, G753E, S757D, 37 90 120 137 A769D, L775A,D801G, A843P, K875T, N892Y Q109R, E229S, T631N, N672D, P752R, G753E,S754E, 37 90 120 138 S757D, A769D, L775A, D801G, K875T, N892Y, K1016TE229S, D458S, A624E, S635E, T649K, I656V, N672D, 37 90 120 138 G753E,S754R, S757D, A769D, L775A, D777K, D801G, K875T, N892Y A190Q, E229S,K360G, D458S, S582K, N672D, G753E, 37 90 120 141 S754E, A769D, L775A,D801G, K875T, N892Y, N1008D S100D, E229S, N440K, S582K, T631N, N672D,G753E, 37 90 120 142 S754E, A769D, L775A, D801G, K875T, N892Y, N1008DS100D, E229S, K360G, D458S, S582K, N672D, G753E, 37 90 120 144 S754E,A769D, L775A, D801G, A843P, K875T, N892Y, N1008D E229S, I234V, A492L,N672D, G753E, S754E, A769D, 37 90 120 145 L775A, D777K, D801G, K875T,N892Y A190Q, E229S, K360G, D458S, S582K, T664K, N672D, 37 90 120 146G753E, S754E, A769D, L775A, D801G, K875T, N892Y, N1008D A190Q, E229S,D458S, T631N, N672D, G753E, S754E, 37 90 120 147 A769D, L775A, D801G,A843P, K875T, N892Y E229S, N440K, S582K, S635E, N672D, G753E, S754E, 3790 120 147 A769D, L775A, D801G, A843P, K875T, N892Y, N1008D E229S,A492L, S635E, T649K, I656V, N672D, P752R, G753E, 37 90 120 147 S754E,A769D, L775A, D801G, K875T, N892Y, N1008D T18D, E229S, S582K, N672D,G753E, S754E, A769D, L775A, 37 90 120 149 D801G, K875T, N892Y, T902FE229S, D458S, S582K, A624E, T631N, N672D, M728V, 37 90 120 149 G753E,S754E, S757D, A769D, L775A, D801G, K875T, N892Y E229S, D458S, T631E,N672D, G753E, S754E, S757D, 37 90 120 151 A769D, L775A, D801G, K875T,N892Y T18D, E229S, N440K, S582K, A624E, S635E, N672D, G753E, 37 90 120152 S754E, S757D, A769D, L775A, D801G, K875T, N892Y E229S, N440K, S582K,A624E, N672D, P752R, G753E, 37 90 120 152 S754E, A769D, L775A, D801G,K875T, N892Y, N1008D E229S, N440K, S582K, A624E, T631N, S635E, N672D, 3790 120 153 G753E, S754E, S757D, A769D, L775A, D801G, K875T, N892Y E229S,N440K, S582K, A624E, S635E, N672D, G738L, 37 90 120 154 G753E, S754E,S757D, A769D, L775A, D801G, K875T, N892Y E229S, I234V, A492L, S582K,N672D, M728V, G753E, 37 90 120 157 S754E, A769D, L775A, D801G, K875T,N892Y E229S, D458S, A492L, T631N, N672D, G753E, S754E, 37 90 120 158S757D, A769D, L775A, D801G, K875T, N892Y A190Q, E229S, I234V, S582K,N672D, G753E, S754E, 37 90 120 158 S757D, A769D, L775A, D801G, K875T,N892Y E229S, N440K, S582K, N672D, P752R, G753E, S754E, 37 90 120 158S757D, A769D, L775A, D801G, K875T, N892Y, N1008D E229S, S635E, T649K,I656V, N672D, G753E, S754R, S757D, 37 90 120 159 A769D, L775A, D801G,A843P, K875T, N892Y A190Q, E229S, N440K, S582K, A624E, S635E, N672D, 3790 120 162 G753E, S754E, S757D, A769D, L775A, D801G, K875T, N892Y S100D,E229S, D458S, K567R, S582K, S635E, N672D, 37 90 120 163 G753E, S754E,A769D, L775A, D801G, K875T, N892Y E229S, S635E, N672D, P752R, G753E,S754E, A769D, 37 90 120 169 L775A, D777K, D801G, K875T, N892Y S100D,A190Q, E229S, K360G, D458S, S582K, N672D, 37 90 120 170 G753E, S754E,A769D, L775A, D801G, K875T, N892Y, N1008D S100D, E229S, K360G, D458S,S582K, N672D, G753E, 37 90 120 171 S754E, A769D, L775A, D801G, K875T,N892Y, N1008D E229S, D458S, S582K, T631N, S635E, N672D, M728V, 37 90 120172 G753E, S754E, S757D, A769D, L775A, D801G, K875T, N892Y S100D, E229S,K360G, D458S, S582K, N672D, G753E, 37 90 120 174 S754E, S757D, A769D,L775A, D801G, A843P, K875T, N892Y, N1008D E229S, I234V, A492L, S582K,N672D, G753E, S754E, 37 90 120 174 A769D, L775A, D801G, K875T, N892YE229S, D458S, S582K, T631E, N672D, G753E, S754E, 37 90 120 175 A769D,L775A, D801G, K875T, N892Y E229S, I234V, A492L, N672D, G753E, S754E,A769D, 37 90 120 176 L775A, D777R, D801G, K875T, N892Y S100D, E229S,K360G, D458S, S582K, N672D, G753E, 37 90 120 179 S754E, S757D, A769D,L775A, D801G, A843P, K875T, N892Y, N1008D E229S, A624E, S635E, T649K,I656V, N672D, G753E, S754R, 37 90 120 185 S757D, A769D, L775A, D777K,D801G, K875T, N892Y S100D, E229S, K360G, D458S, S582K, N672D, G753E, 3790 120 189 S754E, S757D, A769D, L775A, D801G, A843P, K875T, N892Y,T915A, N1008D T18D, E229S, D458S, T631N, N672D, M728V, G753E, 37 90 120191 S754E, S757D, A769D, L775A, D801G, K875T, N892Y S100D, E229S, N440K,S582K, N672D, P752R, G753E, 37 90 120 192 S754E, A769D, L775A, D801G,K875T, N892Y, N1008D T18D, E229S, S582K, N672D, G753E, S754E, P764K,A769D, 37 90 120 202 L775A, D801G, K875T, N892Y E229S, D458S, T631E,N672D, G753E, S754E, A769D, 37 90 120 203 L775A, D777K, D801G, K875T,N892Y E229S, S582K, S635E, T649K, I656V, N672D, P752K, G753E, 37 90 120209 S757D, A769D, L775A, D801G, A843P, K875T, N892Y E229S, A624E, S635E,T649K, I656V, N672D, G738L, 37 90 120 210 G753E, S754R, S757D, A769D,L775A, D777K, D801G, K875T, N892Y A159P, E229S, D458S, T631N, N672D,M728V, G753E, 37 90 120 223 S754E, S757D, A769D, L775A, D801G, K875T,N892Y A190Q, E229S, S582K, N672D, G753E, S754E, A769D, 37 90 120 226L775A, D801G, K875T, N892Y A190Q, E229S, K360G, D458S, S582K, N672D,G753E, 37 90 120 234 S754E, S757D, A769D, L775A, D801G, K875T, N892Y,N1008D

While at least one exemplary embodiment has been presented in theforegoing detailed description, it should be appreciated that a vastnumber of variations exist. It should also be appreciated that theexemplary embodiment or exemplary embodiments are only examples, and arenot intended to limit the scope, applicability, or configuration of thevarious embodiments in any way. Rather, the foregoing detaileddescription will provide those skilled in the art with a convenient roadmap for implementing an exemplary embodiment as contemplated herein. Itbeing understood that various changes may be made in the function andarrangement of elements described in an exemplary embodiment withoutdeparting from the scope of the various embodiments as set forth in theappended claims.

1. A detergent composition comprising a xanthan lyase variant,comprising an alteration at one or more positions in at least onechelator-induced instability region selected from the group of: i)region 3 corresponding to amino acids 731 to 803 of SEQ ID NO:2, ii)region 6 corresponding to amino acids 903 to 1004 of SEQ ID NO: 2, iii)region 1 corresponding to amino acids 154 to 176 of SEQ ID NO: 2, iv)region 2 corresponding to amino acids 614 to 658 of SEQ ID NO: 2, v)region 4 corresponding to amino acids 807 to 846 of SEQ ID NO:2, and vi)region 5 corresponding to amino acids 872 to 885 of SEQ ID NO: 2,wherein said variant has at least about 60% and less than about 100%sequence identity to SEQ ID NO:
 2. 2. The detergent composition of claim1, wherein said variant has at least about 61% sequence identity to SEQID NO:
 2. 3. The detergent composition of claim 1, wherein saidalteration at one or more positions is selected from the group ofalterations in positions: 155, 159, 620, 624, 626, 631, 635, 649, 650,656, 738, 745, 752, 753, 754, 757, 769, 775, 777, 779, 782, 786, 789,792, 796, 799, 801, 819, 824, 843, 875, 903, 911, 912, 915, 919, 921,923, 925, 927, 928, 930, 933, 941, 966, 991 and 998, wherein numberingis according to SEQ ID NO:
 2. 4. The detergent composition of claim 1,wherein said alteration at one or more positions is selected from thegroup of: Y155E, A159P, K620R, A624E, A626Q, T631N, S635E, S635T, T649V,T649K, Q650G, I656V, G738L, K745R, P752R, P752K, G753E, G753Q, G753S,S754E, S754L, S754Q, S754R, S757D, S757P, S757E, A769D, A769T, A769R,L775A, L775F, L775I, L775M, L775Q, L775S, L775Y, D777R, P779V, Y782I,N786K, G789R, K792W, K792Y, K792V, K792A, N796Q, A799H, D801G, K819R,K824R, A843P, K875T, K875E, T903A, T903Q, A911M, A911V, A911S, A912T,A912I, A912Y, T915S, T915V, T915Q, T919D, T919F, T919G, T921R, T921S,T923D, T923H, T925D, T925Q, T925R, T927K, D928W, Y930F, Y930H, Y930L,D933M, G941D, G941E, A966P, N991D and V998K, wherein numbering isaccording to SEQ ID NO:
 2. 5. The detergent composition of claim 1, thexanthan lyase variant further comprising an alteration at one or morepositions in at least one region selected from the group of: vii) region7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, viii) region 8corresponding to amino acids 177 to 613 of SEQ ID NO: 2, ix) region 9corresponding to amino acids 659 to 730 of SEQ ID NO: 2, x) region 10corresponding to amino acids 804 to 806 of SEQ ID NO: 2, xi) region 11corresponding to amino acids 847 to 871 of SEQ ID NO: 2, xii) region 12corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and xiii)region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO:
 2. 6.The detergent composition of claim 5, wherein said alteration at one ormore positions in at least one region selected from the group of regions7, 8, 9, 10, 11, 12 and 13 is an alteration at one or more positionsselected from the group of: 9, 15, 18, 46, 58, 66, 89, 95, 100, 106,109, 183, 188, 190, 203, 204, 221, 229, 234, 238, 240, 242, 243, 257,258, 284, 291, 293, 316, 317, 320, 324, 329, 333, 339, 341, 352, 354,360, 372, 377, 399, 400, 419, 440, 450, 451, 454, 458, 481, 492, 505,533, 567, 568, 576, 578, 579, 582, 664, 672, 703, 722, 726, 727, 728,851, 855, 856, 867, 887, 892, 899, 900, 901, 902, 915, 1008 and 1016,wherein numbering is according to SEQ ID NO:
 2. 7. The detergentcomposition of claim 6, wherein said alteration at one or more positionsin at least one region selected from the group of regions 7, 8, 9, 10,11, 12 and 13 comprises one or more substitutions selected from thegroup of: K9R, N15T, T18D, L46D, A58L, S66H, Q89Y, K95E, S100D, N106Y,Q109R, Q109D, Q109F, Q109K, Q109A, K183Q, K183R, V188I, A190Q, A203P,K204R, A221P, E229N, E229S, E229V, I234V, I238W, I238L, I238M, 1240W,N242S, G243V, Y257W, R258E, R284G, K291R, A293G, A293P, K316R, R317K,K320R, L324Q, K329R, K333R, L339M, I341P, V352I, S354P, K360G, K360R,Q372H, F377Y, N399K, K400R, F419Y, N440K, D450P, K451E, K451R, A454V,D458S, K481R, A492H, A492L, T5051, L533I, K567R, G568A, S578K, S578N,S578R, S579R, S579K, S582K, T664K, N672D, I703L, I722F, P726Q, T727P,M728V, S851F, K855R, E856D, P867S, K887R, N892Y, N892W, N892F, G899S,1900G, D901A, T902F, N1008D and K1016T, wherein numbering is accordingto SEQ ID NO:
 2. 8. The detergent composition of claim 6, wherein thexanthan lyase variant comprises an alteration at one or more positionsselected from the group of positions 624, 631, 635, 649, 656, 738, 752,753, 754, 757, 769, 775, 777, 800, 801, 843, 875, 911 and 915, and analteration at one or more positions selected from the group of positions89, 100, 190, 229, 234, 352, 360, 399, 440, 458, 492, 567, 582, 664,672, 703, 728, 892, 1008 and 1016 of SEQ ID NO:
 2. 9. The detergentcomposition of claim 8, wherein the xanthan lyase variant comprises oneor more substitutions selected from the group of Q89Y, S100D, A190Q,E229S, I234V, V352I, K360G, N399K, N440K, D458S, A492H, A492L, K567R,S582K, T664K, N672D, I703L, M728V, N892Y N1008D and K1016T, and one ormore substitutions selected from the group of A624E, T631N, S635E,T649K, I656V, G738L, P752K, P752R, G753E, S754E, S754R, S757D, A769D,L775A, D777R, V800P, D801G, A843P, K875T, A911V and T915A.
 10. Thedetergent composition of claim 1, wherein said xanthan lyase variant hasan improved stability in a detergent composition compared to a parentxanthan lyase.
 11. The detergent composition of claim 1, wherein saidxanthan lyase variant has a half-life improvement factor (HIF) of >about 1.0, relative to a parent xanthan lyase.
 12. The detergentcomposition comprising at least one xanthan lyase variant of claim 1,wherein said composition further comprises one or more detergentcomponents.
 13. A method for degrading xanthan gum comprising the stepof applying the detergent composition of claim 1 to a xanthan gumdisposed on a surface of a textile or on a hard surface.
 14. Thedetergent composition of claim 1, wherein said variant has at leastabout 99% sequence identity to SEQ ID NO:
 2. 15. The detergentcomposition of claim 1, wherein said variant has at least about 99%sequence identity to SEQ ID NO: 2; wherein said alteration at one ormore positions is selected from the group of alterations in positions:155, 159, 620, 624, 626, 631, 635, 649, 650, 656, 738, 745, 752, 753,754, 757, 769, 775, 777, 779, 782, 786, 789, 792, 796, 799, 801, 819,824, 843, 875, 903, 911, 912, 915, 919, 921, 923, 925, 927, 928, 930,933, 941, 966, 991 and 998, wherein numbering is according to SEQ ID NO:2; wherein said alteration at one or more positions is selected from thegroup of: Y155E, A159P, K620R, A624E, A626Q, T631N, S635E, S635T, T649V,T649K, Q650G, I656V, G738L, K745R, P752R, P752K, G753E, G753Q, G753S,S754E, S754L, S754Q, S754R, S757D, S757P, S757E, A769D, A769T, A769R,L775A, L775F, L775I, L775M, L775Q, L775S, L775Y, D777R, P779V, Y782I,N786K, G789R, K792W, K792Y, K792V, K792A, N796Q, A799H, D801G, K819R,K824R, A843P, K875T, K875E, T903A, T903Q, A911M, A911V, A911S, A912T,A912I, A912Y, T915S, T915V, T915Q, T919D, T919F, T919G, T921R, T921S,T923D, T923H, T925D, T925Q, T925R, T927K, D928W, Y930F, Y930H, Y930L,D933M, G941D, G941E, A966P, N991D and V998K, wherein numbering isaccording to SEQ ID NO: 2; wherein the xanthan lyase variant furthercomprises an alteration at one or more positions in at least one regionselected from the group of: vii) region 7 corresponding to amino acids 1to 153 of SEQ ID NO: 2, viii) region 8 corresponding to amino acids 177to 613 of SEQ ID NO: 2, ix) region 9 corresponding to amino acids 659 to730 of SEQ ID NO: 2, x) region 10 corresponding to amino acids 804 to806 of SEQ ID NO: 2, xi) region 11 corresponding to amino acids 847 to871 of SEQ ID NO: 2, xii) region 12 corresponding to amino acids 886 to902 of SEQ ID NO: 2, and xiii) region 13 corresponding to amino acids1005 to 1037 of SEQ ID NO: 2; wherein said alteration at one or morepositions in at least one region selected from the group of regions 7,8, 9, 10, 11, 12 and 13 is an alteration at one or more positionsselected from the group of: 9, 15, 18, 46, 58, 66, 89, 95, 100, 106,109, 183, 188, 190, 203, 204, 221, 229, 234, 238, 240, 242, 243, 257,258, 284, 291, 293, 316, 317, 320, 324, 329, 333, 339, 341, 352, 354,360, 372, 377, 399, 400, 419, 440, 450, 451, 454, 458, 481, 492, 505,533, 567, 568, 576, 578, 579, 582, 664, 672, 703, 722, 726, 727, 728,851, 855, 856, 867, 887, 892, 899, 900, 901, 902, 915, 1008 and 1016,wherein numbering is according to SEQ ID NO: 2; and wherein saidalteration at one or more positions in at least one region selected fromthe group of regions 7, 8, 9, 10, 11, 12 and 13 comprises one or moresubstitutions selected from the group of: K9R, N15T, T18D, L46D, A58L,S66H, Q89Y, K95E, S100D, N106Y, Q109R, Q109D, Q109F, Q109K, Q109A,K183Q, K183R, V188I, A190Q, A203P, K204R, A221P, E229N, E229S, E229V,I234V, I238W, I238L, I238M, 1240W, N242S, G243V, Y257W, R258E, R284G,K291R, A293G, A293P, K316R, R317K, K320R, L324Q, K329R, K333R, L339M,I341P, V352I, S354P, K360G, K360R, Q372H, F377Y, N399K, K400R, F419Y,N440K, D450P, K451E, K451R, A454V, D458S, K481R, A492H, A492L, T5051,L533I, K567R, G568A, S578K, S578N, S578R, S579R, S579K, S582K, T664K,N672D, I703L, I722F, P726Q, T727P, M728V, S851F, K855R, E856D, P867S,K887R, N892Y, N892W, N892F, G899S, 1900G, D901A, T902F, N1008D andK1016T, wherein numbering is according to SEQ ID NO:
 2. 16. Thedetergent composition of claim 15, wherein said xanthan lyase varianthas an improved stability in a detergent composition compared to aparent xanthan lyase with SEQ ID NO: 2; wherein said xanthan lyasevariant has a half-life improvement factor (HIF) of at least 2.0relative to the parent xanthan lyase; and wherein said compositionfurther comprises one or more detergent components.
 17. The detergentcomposition of claim 1, wherein said variant has at least about 99%sequence identity to SEQ ID NO: 2; wherein said alteration at one ormore positions is selected from the group of alterations in positions:155, 159, 620, 624, 626, 631, 635, 649, 650, 656, 738, 745, 752, 753,754, 757, 769, 775, 777, 779, 782, 786, 789, 792, 796, 799, 801, 819,824, 843, 875, 903, 911, 912, 915, 919, 921, 923, 925, 927, 928, 930,933, 941, 966, 991 and 998, wherein numbering is according to SEQ ID NO:2; wherein said alteration at one or more positions is selected from thegroup of: Y155E, A159P, K620R, A624E, A626Q, T631N, S635E, S635T, T649V,T649K, Q650G, I656V, G738L, K745R, P752R, P752K, G753E, G753Q, G753S,S754E, S754L, S754Q, S754R, S757D, S757P, S757E, A769D, A769T, A769R,L775A, L775F, L775I, L775M, L775Q, L775S, L775Y, D777R, P779V, Y782I,N786K, G789R, K792W, K792Y, K792V, K792A, N796Q, A799H, D801G, K819R,K824R, A843P, K875T, K875E, T903A, T903Q, A911M, A911V, A911S, A912T,A912I, A912Y, T915S, T915V, T915Q, T919D, T919F, T919G, T921R, T921S,T923D, T923H, T925D, T925Q, T925R, T927K, D928W, Y930F, Y930H, Y930L,D933M, G941D, G941E, A966P, N991D and V998K, wherein numbering isaccording to SEQ ID NO: 2; wherein the xanthan lyase variant furthercomprises an alteration at one or more positions in at least one regionselected from the group of: vii) region 7 corresponding to amino acids 1to 153 of SEQ ID NO: 2, viii) region 8 corresponding to amino acids 177to 613 of SEQ ID NO: 2, ix) region 9 corresponding to amino acids 659 to730 of SEQ ID NO: 2, x) region 10 corresponding to amino acids 804 to806 of SEQ ID NO: 2, xi) region 11 corresponding to amino acids 847 to871 of SEQ ID NO: 2, xii) region 12 corresponding to amino acids 886 to902 of SEQ ID NO: 2, and xiii) region 13 corresponding to amino acids1005 to 1037 of SEQ ID NO: 2; wherein said alteration at one or morepositions in at least one region selected from the group of regions 7,8, 9, 10, 11, 12 and 13 is an alteration at one or more positionsselected from the group of: 9, 15, 18, 46, 58, 66, 89, 95, 100, 106,109, 183, 188, 190, 203, 204, 221, 229, 234, 238, 240, 242, 243, 257,258, 284, 291, 293, 316, 317, 320, 324, 329, 333, 339, 341, 352, 354,360, 372, 377, 399, 400, 419, 440, 450, 451, 454, 458, 481, 492, 505,533, 567, 568, 576, 578, 579, 582, 664, 672, 703, 722, 726, 727, 728,851, 855, 856, 867, 887, 892, 899, 900, 901, 902, 915, 1008 and 1016,wherein numbering is according to SEQ ID NO: 2; wherein the xanthanlyase variant comprises an alteration at one or more positions selectedfrom the group of positions 624, 631, 635, 649, 656, 738, 752, 753, 754,757, 769, 775, 777, 800, 801, 843, 875, 911 and 915, and an alterationat one or more positions selected from the group of positions 89, 100,190, 229, 234, 352, 360, 399, 440, 458, 492, 567, 582, 664, 672, 703,728, 892, 1008 and 1016 of SEQ ID NO: 2; and wherein the xanthan lyasevariant comprises one or more substitutions selected from the group ofQ89Y, S100D, A190Q, E229S, I234V, V352I, K360G, N399K, N440K, D458S,A492H, A492L, K567R, S582K, T664K, N672D, I703L, M728V, N892Y N1008D andK1016T, and one or more substitutions selected from the group of A624E,T631N, S635E, T649K, I656V, G738L, P752K, P752R, G753E, S754E, S754R,S757D, A769D, L775A, D777R, V800P, D801G, A843P, K875T, A911V and T915A.18. The detergent composition of claim 17, wherein said xanthan lyasevariant has an improved stability in a detergent composition compared toa parent xanthan lyase with SEQ ID NO: 2; wherein said xanthan lyasevariant has a half-life improvement factor (HIF) of at least 2.0relative to the parent xanthan lyase; and wherein said compositionfurther comprises one or more detergent components.